Recombinant herpes simplex virus having an expression cassette capable of expressing a fusion protein formed by a cancer cell targeting region and an extracellular domain of hvem and uses thereof

Abstract

This invention discloses a recombinant herpes simplex virus having an expression cassette capable of expressing a fusion protein formed by a cancer cell targeting region and an extracellular domain of HVEM; and its use. When the recombinant HSV infects target cells, i.e., cancer cells, and enters the target cells, as the HSV proliferates, the adaptor protein of the fusion protein is expressed in the cells, causing it to be released extracellularly along with the proliferating HSV viral particles via cell lysis, or even before the viral particles are released via cell lysis if the adaptor protein includes a leader sequence. The fusion protein released extracellularly induces the HSV viral particles to infect surrounding cancer cells expressing target molecules recognized by the cancer cell targeting region of the adaptor protein, or enhances infection efficiency.

Description

Invention Field

[0001] This invention relates to a recombinant herpes simplex virus having an expression cassette capable of expressing a fusion protein of a cancer cell targeting domain and an HVEM extracellular domain; and its use. Background Technology

[0002] To date, surgical treatments, chemotherapy, and radiotherapy have been widely used in cancer treatment. However, most of these therapies have side effects, incomplete treatment outcomes, and problems such as cancer recurrence and metastasis. Therefore, there is a continuous need to develop new and effective cancer therapies. In recent years, anti-cancer immunotherapy has developed rapidly, such as oncolytic viruses and chimeric antigen receptor T (CAR-T) cell therapy.

[0003] In anticancer immunotherapy, oncolytic viruses are viruses characterized by manipulating the genes of live viruses and selectively proliferating within cancer cells to lyse them, while their proliferation in normal cells is restricted. The viruses released through the lysis of cancer cells can continuously infect surrounding cancer cells, thus providing a continuous and synergistic therapeutic effect. Furthermore, oncolytic viruses can stimulate the body's immune response by releasing immunogenic tumor antigens during the lysis of cancer cells, thereby enhancing their anticancer effect. Moreover, this anticancer effect can be further enhanced through artificial manipulation to express cytokines and chemokines.

[0004] Currently developed oncolytic viruses can be classified into 10 or more types, including adenovirus, herpes simplex virus (HSV), and vaccinia virus. HSV, in particular, is an enveloped icosahedral viral particle containing a 152kb linear double-stranded DNA, and is divided into HSV-1 and HSV-2 types. HSV possesses many non-essential genes and a large genome size, facilitating the manipulation or transport of external genes. Furthermore, it has a short replication cycle and high infection efficiency. Ideally, it can exhibit enhanced cancer cell targeting efficiency through simple manipulation of glycoproteins associated with cell attachment and infection.

[0005] T-VEC (talimogene laherparepvec, product name: Imlygic) was approved by the US FDA in October 2015. It is an anticancer drug (oncolytic virus therapy) using HSV-1 to treat malignant melanoma. T-VEC is an attenuated HSV-1 virus with the ICP34.5 and ICP47 genes deleted, thus reducing its pathogenicity, and it also expresses GM-CSF (granulocyte-macrophage colony-stimulating factor), thereby promoting the human immune response. However, T-VEC has limitations because viral replication is restricted due to the loss of some genes, resulting in lower therapeutic efficacy.

[0006] HSV is an enveloped virus that enters cells through the complex interactions of gD, gB, gH / gL, and gC glycoproteins present in the envelope. First, when gB and gC are attached to 3-OS HS (3-O-sulfated heparan sulfate) on the cell surface, gD binds to at least one of the following cell receptors: HVEM (herpesvirus entry mediator, HveA), cohesin-1 (HveC), and cohesin-2 (HveB), thus inducing viral fusion with the cell membrane, thereby allowing HSV to enter the cell (Hiroaki Uchida et al., Generation of Herpesvirus Entry Mediator (HVEM)-restricted Herpes Simplex Virus Type 1 Mutant Viruses: Resistance of HVEM-expressing Cells and Identification of Mutations That Rescue). nectin-1Recognition). J.Virol. April 2009; 83(7):2951-61.

[0007] Among the cell receptors of HSV, HVEM belongs to the tumor necrosis factor receptor protein family (TNFR family), and is mainly expressed in T / B lymphocytes, macrophages and DC cells, sensory neurons and mucosal epithelial cells (Shui JW, Kronenberg M. 2013. Gut Microbes 4(2):146-151). However, it is known to be highly expressed in various tumor tissues such as B / T lymphoma, melanoma, colorectal cancer, hepatocellular carcinoma, breast cancer, ovarian serous adenocarcinoma, renal clear cell carcinoma and glioblastoma (Pasero C. et al., Curr. Opin. Pharmacol. 2012. 2(4):478-85; Malissen N. et al., ONCOIMMUNOLOGY 2019, VOL. 8(12):e1665976). HVEM has four CRDs (cysteine ​​enrichment domains), which are characteristic of the TNFR family. Two of the four CRDs are linked to the gD of HSV, thus inducing HSV-1 and HSV-2 to enter the cell (Sarah A Connolly et al., "Structure-based Analysis of the Herpes Simplex Virus Glycoprotein D Binding Site Present on Herpesvirus EntryMediator HveA (HVEM)". J. Virol. November 2002; 76(21):10894-904).

[0008] As reported by the inventors of this invention, a bispecific fusion protein (CEAscFv-HveA) of CEA (carcinoembryonic antigen) scFv (single-chain variable fragment) and HVEM (an HSV cell surface receptor) extracellular domain was manufactured, and when CEA-expressing cell lines were treated with the bispecific fusion protein and HSV, the fusion protein acted as an adaptor protein to induce HSV to target and infect the corresponding cell lines (Korean Patent No. 10-0937774 and US Patent No. 8318662).

[0009] When the gene encoding a fusion protein (CEAscFv-HveA) used as an adaptor protein is expressibly inserted into the HSV genome to express the fusion protein in HSV-infected cells, the fusion protein is determined to induce HSV to target and infect cell lines expressing CEA. Moreover, it is not the scFv of CEA, but the scFv of HER2 (human epidermal growth factor receptor 2) that induces the targeting and infection of HER2-expressing cell lines equally with the fusion protein of the extracellular domain of HVEM (HER2scFv-HveA), thus the present invention was developed. Summary of the Invention

[0010] This article discloses a recombinant HSV containing an expression cassette capable of expressing an adaptor protein, which is a fusion protein of a cancer cell targeting domain and an extracellular domain of HVEM.

[0011] In addition, this article also discloses a pharmaceutical composition for anticancer treatment, which contains the aforementioned recombinant HSV as an active ingredient.

[0012] In addition, this article also discloses a method for treating or preventing cancer (tumor) comprising administering to a subject, such as a patient, an effective amount of a pharmaceutical composition containing the aforementioned recombinant HSV.

[0013] Other or specific aspects of the invention will be provided below. Detailed Implementation

[0014] This invention relates to a recombinant HSV (herpes simplex virus) capable of expressing an adaptor protein, which is a fusion protein of a cancer cell targeting domain (which may be used interchangeably with the cancer targeting domain herein, or may be represented as a ligand, depending on the context of this specification) and an HVEM extracellular domain.

[0015] When the recombinant HSV of this invention infects target cells, i.e., cancer cells, and enters the target cells, the HSV proliferates, and the adaptor protein, as a fusion protein, is expressed in the cells. This adaptor protein is released extracellularly along with the proliferating HSV viral particles after cell lysis, or continuously before the viral particles are released due to cell lysis, provided the adaptor protein contains a leader sequence. The extracellular adaptor protein is used to induce the HSV viral particles to infect surrounding cancer cells expressing target molecules recognized by the cancer cell targeting domain of the adaptor protein, or to enhance infection efficiency.

[0016] Generally speaking, recombinant HSVs are HSVs that, compared to wild-type HSVs, are genetically manipulated by introducing artificial mutations (through the deletion, substitution, or insertion of some nucleic acid sequences) to lose or alter certain functions or express target proteins of interest. In this invention, the recombinant HSV is an HSV that can express adaptor proteins in cancer cells infected with it by introducing (i.e., inserting) a cassette expressing adaptor proteins (i.e., a construct that operatively links the adaptor protein gene with a promoter sequence and a polyadenylation signal sequence that enable its expression) into the HSV genome without inhibiting HSV proliferation. Recombinant virus production techniques, such as viral gene manipulation and viral particle generation, are well-known in the field. See also [Sandri-Goldin RM et al., Alpha Herpesviruses: Molecular and Cellular Biology, Caister Academic Press, 2006] and [Robin H. H. Achmann, Herpes simplex virus-based vectors, Int J Exp Pathol. August 2004; 85(4):177-190]. All references cited in this specification, including those above, are considered part of this specification.

[0017] Specifically, in addition to being manipulated to express adaptor proteins, the recombinant HSV of the present invention can also be manipulated to enter cells only via the HVEM receptor as an entry receptor, without via cohesin-1. In the following examples of the present invention, the sequence of the HSV envelope glycoprotein gD is manipulated to allow HSV to enter cells only via the HVEM receptor. Specifically, arginine (R) at position 222 and phenylalanine (F) at position 223 of gD are substituted with asparagine (N) and isoleucine (I), respectively, thereby altering the function of gD. This alters the function of recombinant HSV, which can only enter host cells via the HVEM (HveA) receptor (Hiroaki Uchida et al., Generation of Herpes simplex virus type 1 mutant viruses restricted by herpes simplex virus entry mediator (HVEM): Resistance of HVEM-expressing Cells and Identification of Mutations That Rescue nectin-1 Recognition). J. Virol. April 2009; 83(7):2951-61). HVEM (HveA) receptors are known to be highly expressed in various tumor tissues such as B / T lymphoma and melanoma (Pasero C. et al., Curr. Opin. Pharmacol. 2012. 2(4): 478-85; Malissen N. et al., ONCOIMMUNOLOGY 2019, VOL. 8(12): e1665976).

[0018] Furthermore, the recombinant HSV of the present invention can be mutated such that non-essential genes that are not required for HSV proliferation (i.e., survival and replication) are missing or do not exhibit the function of these genes (i.e., transcription or translation is interrupted). Specific examples of non-essential genes may include the UL3 gene (e.g., GenBank accession number AFE62830.1), UL4 gene (e.g., GenBank accession number AFE62831.1), UL14 gene (e.g., GenBank accession number AFE62841.1), UL16 gene (e.g., GenBank accession number AFE62843.1), UL21 gene (e.g., GenBank accession number AFE62848.1), UL24 gene (e.g., GenBank accession number AFE62851.1), UL31 gene (e.g., GenBank accession number AFE62859.1), UL32 gene (e.g., GenBank accession number AFE62860.1), US3 gene (e.g., GenBank accession number AFE62891.1), and UL51 gene (e.g., GenBank accession number A...). FE62880.1), UL55 gene (e.g., GenBank accession number AFE62884.1), UL56 gene (e.g., GenBank accession number AFE62885.1), US2 gene (e.g., GenBank accession number AFE62890.1), US12 gene (e.g., GenBank accession number AFE62901.1; ICP47 gene), LAT gene (e.g., GenBank accession number JQ673480.1), gB gene (e.g., the sequence between 52996 and 55710 of GenBank accession number GU734771.1), gL gene (e.g., GenBank accession number AFE62828.1), gH gene (e.g., GenBank accession number AFE62849.1), gD gene (e.g., GenBank accession number AFE62894.1), etc.

[0019] For specific information on non-essential genes of HSV, please refer to the following papers: [DM Knipe and PM Howley (eds.) Fields virology (Vol. 2) Lippincott Williams & Wilkins, Philadelphia, Pa. 2001. pp. 2399-2460], [Subak-Sharpe JH, Dargan DJ "HSV molecular biology: general aspects of herpes simplex virus molecular biology" Virus Genes, 1998, 16(3): 239-251], and [Travis J. Taylor and David M. Knipe, "Proteomics of Herpes Simplex Virus Replication Compartments: Association of Cellular DNA Replication, Repair, Recombination, and Chromatin-Remodeling Proteins with ICP8"]. ICP8), J.Virol. June 2004; 78(11):5856-5866, etc.

[0020] The recombinant HSV of the present invention can be a recombinant HSV-1 virus, a recombinant HSV-2 virus, or an HSV-1 / HSV-2 chimeric virus (i.e., a recombinant HSV whose genome contains both DNA derived from HSV-1 and DNA derived from HSV-2), preferably a recombinant HSV-1 virus, and more preferably a recombinant HSV-1 derived from the HSV-1KOS virus strain. The HSV-1KOS virus strain can be obtained from ATCC (catalog number VR-1493TM), and the complete genome sequence of the virus strain is fully analyzed and represented in GenBank accession number JQ673480.1 (Stuart J. Macdonald et al., Genome Sequence of Herpes Simplex Virus 1 Strain KOS., J. Virol. 2012 June; 86(11):6371-2).

[0021] The HSV-1 viral genome consists of 152 kb of double-stranded linear DNA, encoding a total of 84 genes. It comprises two interconnected segments: a long segment (L region) and a short segment (S region). The long segment (L region) accounts for approximately 82% of the genome, and the short segment (S region) accounts for approximately 18%. The long and short segments are linked by two IRLs (intermediate inverted repeats) serving as boundary regions, and each segment has a TRL (terminal inverted repeat) at its end. The L region (UL) contains 56 genes (UL1-UL56) and 10 genes (UL8.5, 9.5, 10.5, 12.5, 15.5, 20.5, 26.5, 27.5, 43.5, 49.5). The S region (US) contains 12 genes (US1-US12) and 2 genes (US1.5, 8.5). The two IRLs serving as boundary regions contain 4 genes (ICP4, ICP34.5, ICP0, and LAT).

[0022] In this invention, the cassette for expressing the adaptor protein is configured such that the adaptor protein gene is operatively linked to a promoter sequence that enables the expression of the adaptor protein gene and a polyadenylation signal sequence that serves as a transcription termination signal sequence. Here, "operatively linked" means a link that enables the expressed adaptor protein gene to be transcribed and / or translated. For example, the promoter is operatively linked to the adaptor protein gene when any promoter affects the transcription of the adaptedor protein gene linked to it.

[0023] Typically, a promoter is a nucleic acid sequence that controls the transcription of one or more genes. It is located upstream (5' side) of the transcription start site and includes a DNA-dependent RNA polymerase binding site, a transcription start site, a transcription factor binding site, etc. In the case of eukaryotic origin, the promoter includes the TATA box, upstream of the transcription start site (relative to the transcription start site (+1), usually located at position -20 to -30); the CAAT box (relative to the transcription start site, usually located at position -75); an enhancer; a transcription factor binding site, etc.

[0024] All constitutive promoters (which always induce gene expression), inducible promoters (which induce target gene expression in response to specific external stimuli), tissue-specific promoters (which induce gene expression in specific tissues or cells), non-tissue-specific promoters (which induce gene expression in all tissues or cells), endogenous promoters (derived from virus-infected cells), and exogenous promoters (derived from cells other than virus-infected cells) can be used, provided that the promoter can express the target gene it is linked to. Many promoters are known in the art, and appropriate promoters can be selectively used among them. For example, useful promoters include the CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, HSV (herpes simplex virus) TK (thymidine kinase) promoter, adenovirus late promoter, vaccinia virus 75K promoter, SV40 promoter, metallothionein promoter, CD45 promoter (hematopoietic stem cell-specific promoter), CD14 promoter (monocyte-specific promoter), and cancer cell-specific promoters (tumor-specific promoters), such as survivin, midkine, TERT, CXCR4, etc. Specifically, when using cancer cell-specific promoters, only the expression of adaptor proteins in cancer cells is induced, thereby inhibiting the expression of adaptor proteins in normal cells, thus improving the safety of the recombinant HSV of the present invention.

[0025] In addition to the promoter, the cassette expressing the adaptor protein is also configured to include a transcription termination signal sequence, which is a sequence used as a poly(A) addition signal (polyadenylation signal) to improve the integrity and efficiency of transcription. Many transcription termination signal sequences are known in the art, and appropriate sequences can be selectively used, such as the SV40 transcription termination signal sequence, the HSV TK (herpes simplex virus thymidine kinase) transcription termination signal sequence, and so on.

[0026] A cassette expressing an adaptor protein can be expressibly inserted into the HSV genome without inhibiting HSV proliferation. This insertion can occur in the absence of deletions in the HSV genome, or it can be inserted into a locus where some or all of the non-essential genes in the HSV genome are missing. When inserting the cassette expressing the adaptor protein in the absence of deletions in the HSV genome, it can be inserted between genes. Preferred examples of insertion loci include loci between UL3 and UL4 genes, between UL26 and UL27 genes, between UL37 and UL38 genes, between UL48 and UL49 genes, between UL53 and UL54 genes, and between US1 and US2 genes, etc.

[0027] When a cassette expressing an adaptor protein is inserted into a locus that lacks some or all of the non-essential genes in the HSV genome, the missing non-essential genes can be any non-essential genes as illustrated above.

[0028] In the recombinant HSV of the present invention, the cancer cell targeting domain of the adaptor protein is a site that specifically recognizes and binds to the target molecules of cancer cells that are target cells. The target molecules recognized by the cancer cell targeting domain are any antigens or receptors present on the surface of cancer cells.

[0029] The antigen or receptor is preferably an antigen or receptor expressed only in cancer cells or overexpressed in cancer cells compared to normal cells. Examples of antigens or receptors may include, for example, the following target molecules: EGFRvIII (epidermal growth factor receptor variant III), expressed in glioblastoma; EGFR (epidermal growth factor receptor), overexpressed in undifferentiated thyroid cancer, breast cancer, lung cancer, glioma, etc.; metastin receptor, overexpressed in papillary thyroid cancer, etc.; ErbB-based receptor tyrosine kinase, overexpressed in breast cancer, etc.; HER2 (human epidermal growth factor receptor 2), in breast cancer, bladder cancer, gallbladder cancer, cholangiocarcinoma, esophagogastric junction cancer, etc. Overexpression in: Tyrosine kinase-18 receptor (c-Kit), overexpressed in sarcomatoid renal cell carcinoma, etc.; HGF receptor c-Met, overexpressed in esophageal adenocarcinoma, etc.; CXCR4 or CCR7, overexpressed in breast cancer, etc.; Endothelin-A receptor, overexpressed in prostate cancer; PPAR-δ (peroxisome proliferator-activated receptor δ), overexpressed in rectal cancer, etc.; PDGFR-α (platelet-derived growth factor receptor α), overexpressed in ovarian cancer, etc.; CD133, overexpressed in liver cancer, multiple myeloma, etc.; CEA (carcinoembryonic antigen), overexpressed in lung cancer, etc. Overexpressed in colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, rectal cancer, colon cancer, medullary thyroid carcinoma, etc.; EpCAM (epithelial cell adhesion molecule) is overexpressed in liver cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, etc.; GD2 (bisialoylganglioside) is overexpressed in neuroblastoma, etc.; GPC3 (phosphatidylinositol proteoglycan 3) is overexpressed in hepatocellular carcinoma, etc.; PSMA (prostate-specific membrane antigen) is overexpressed in prostate cancer, etc.; TAG-72 (tumor-associated glycoprotein 72) is overexpressed in ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, etc. Overexpression of various cancers is observed in various cancers, including: GD3 (bisialidylganglioside), overexpressed in melanoma; HLA-DR (human leukocyte antigen-DR), overexpressed in hematologic malignancies and solid tumors; MUC1 (mucin 1), overexpressed in advanced solid tumors; NY-ESO-1 (New York esophageal squamous cell carcinoma 1), overexpressed in advanced non-small cell lung cancer; LMP1 (latent membrane protein 1), overexpressed in nasopharyngeal tumors; TRAILR2 (tumor necrosis factor-associated lysis-inducing ligand receptor), overexpressed in lung cancer, non-Hodgkin's lymphoma, ovarian cancer, colon cancer, colorectal cancer, and pancreatic cancer; and VEGFR2 (vascular endothelial growth factor receptor 2) and HGFR (hepatocyte growth factor receptor), overexpressed in hepatocellular carcinoma. Furthermore, surface antigens of cancer stem cells, such as CD44 and CD166, can also be target molecules.Many target molecules are known in this field to be overexpressed in cancer cells compared to normal cells. In addition to the examples listed above, other target molecules can be found in the following papers: [Anne T Collins et al., Prospective Identification of Tumorigenic Prostate Cancer Stem Cells. Cancer Res. 1 Dec 2005; 65(23):10946-51], [Chenwei Li et al., Identification of Pancreatic Cancer Stem Cells. Cancer Res. 1 Feb 2007; 67(3):1030-7], [Shuo Ma et al., Current Progress in CAR-T Cell Therapy for Solid Tumors. Int. J. Biol. Sci. 7 Sep 2019; 15(12):2548-2560], [Dhaval...] S. Sanchala et al., "Oncolytic Herpes Simplex Viral Therapy: A Stride Toward Selective Targeting of Cancer Cells." Front Pharmacol. 2017 May 16; 8:270, etc.

[0030] Specifically, in this invention, the target molecule is preferably HER2 or CEA.

[0031] The target cells targeted by the adaptor protein of the recombinant HSV of the present invention are any cancer cells that have the cancer cell targeting domain of the adaptor protein of the present invention and are targeted by the target molecules. Cancer cells can be any type of cancer, such as esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, melanoma, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, multiple myeloma, blood cancer, etc.

[0032] In this invention, besides complete antibodies capable of specifically binding to target molecules, the cell-targeting domain of adaptor proteins can also be antibody derivatives or antibody analogs. Antibody derivatives are fragments of complete antibodies that include at least one variable region capable of specifically binding to target molecules; or they can be modified antibodies. Examples of antibody derivatives can include antibody fragments such as Fab, scFv, Fv, VhH, VH, VL, etc.; multivalent or multispecific modified antibodies such as Fab2, Fab3, microantibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, dual scFv, etc.; and so on. Antibody analogs are artificial peptides or polypeptides that, like antibodies, have the ability to specifically bind to target molecules, but differ structurally from antibodies, generally having a lower molecular weight than antibodies. Examples of antibody analogs may include ABD, Adhiron, affibody, affilin, affimer, alphabody, anticarrier, armadillo repeat, centyrin, DARPin, fynomer, Kunitz region, pronectin, and repeatbody.

[0033] A considerable number of papers have been published in this field on antibodies, antibody derivatives, antibody analogs, and their production. Examples of these papers include: [Renate Kunert and David Reinhart, Advances in recombinant antibody manufacturing. Appl. Microbiol. Biotechnol. 2016 Apr; 100(8):3451-61], [Holliger P, Hudson P.J., Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 2005 Sep; 23(9):1126-36], and [Xiaowen Yu et al., Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis. Annual Review of Analytical Sciences]. [Chemistry, 2017, 10: 293-320], paper [Abdul Rasheed Baloch et al., "Antibodymimetics: promising complementary agents to animal-sourced antibodies", Critical Reviews in Biotechnology, 2016, 36: 268-275], etc.

[0034] In this invention, the cell-targeting domain of the adaptor protein is preferably an scFv (single-chain variable fragment). scFv refers to a single-chain antibody in which the heavy chain variable region (VH) and light chain variable region (VL) of an immunoglobulin are linked via a short linker peptide. In scFv, the C-terminus of VH is linked to the N-terminus of VL, or vice versa. In scFv, the linker peptide can be of any length and sequence, as long as it does not interfere with the inherent three-dimensional structure of the heavy and light chains and allows them to be spatially adjacent, thereby possessing the ability to specifically bind to the target molecule. Considering flexibility, solubility, resistance to protein hydrolysis, etc., the linker is preferably composed of at least one amino acid selected from, for example, Ser, Gly, Ala, Thr, etc., and its length is 1-30 amino acids, preferably 3-25 amino acids, and more preferably 8-20 amino acids.

[0035] In this invention, the target molecules targeted by the scFv are HER2 or CEA. Specifically, the scFv targeting HER2 is preferably configured such that the VH of SEQ ID NO:1 and the VL of SEQ ID NO:2 are linked via a linker peptide in the order of VH, linker peptide, and VL (i.e., the C-terminus of VH is linked to the N-terminus of VL via a linker peptide), and the scFv targeting CEA is preferably configured such that the VL of SEQ ID NO:3 and the VH of SEQ ID NO:4 are linked via a linker peptide in the order of VL, linker peptide, and VH (i.e., the C-terminus of VL is linked to the N-terminus of VH via a linker peptide). Here, the linker peptide of the scFv targeting HER2 preferably contains the amino acid sequence of SEQ ID NO:5, and the linker peptide of the scFv targeting CEA preferably contains the amino acid sequence of SEQ ID NO:6.

[0036] In this invention, except for HveA82 of SEQ ID NO:7 (the HveA82 sequence containing the leader sequence is represented in SEQ ID NO:8) used in the following examples, the extracellular domain of HVEM may be HveA87 of SEQ ID NO:9 (the HveA87 sequence containing the leader sequence is represented in SEQ ID NO:10), HveA102 of SEQ ID NO:11 (the HveA102 sequence containing the leader sequence is represented in SEQ ID NO:12), or HveA107 of SEQ ID NO:13 (the HveA107 sequence containing the leader sequence is represented in SEQ ID NO:14), as disclosed in Korean Patent No. 10-0937774 and U.S. Patent No. 8318662 (these documents are considered part of this specification). As demonstrated in Korean Patent No. 10-0937774, HveA87, HveA102 and HveA107 have 5, 20 and 25 more amino acids than HveA82, respectively, and all of these can be used as HSV receptors for adaptor proteins.

[0037] In this invention, the linker sequence can be inserted between the cancer cell targeting domain and the HVEM extracellular domain, and the linker sequence can be a linker of any length and any sequence, as long as it does not inhibit the function of each domain of the adaptor protein. Preferably, the linker sequence contains at least one amino acid selected from the four amino acids Ser, Gly, Ala, and Thr, and its length can be 1-30 amino acids, preferably 3-25 amino acids, and more preferably 8-20 amino acids.

[0038] The adaptor protein of the present invention can also be configured in the order of NH2 / cancer cell targeting domain / HVEM extracellular domain / COOH or the reverse order. When the linker peptide is inserted in the middle, the adaptor protein can be configured in the order of NH2 / cancer cell targeting domain / linker peptide / HVEM extracellular domain / COOH or the reverse order.

[0039] The adaptor protein of the present invention can be configured such that a leader sequence is also attached to its N-terminus, specifically, in an adaptor protein conformation of the sequence NH2 / cancer cell targeting domain / connector peptide / HVEM extracellular domain / COOH, attached to the N-terminus of the cancer cell targeting domain (the N-terminus of VH or VL when using scFv); or in an adaptor protein conformation of the sequence NH2 / HVEM extracellular domain / connector peptide / cancer cell targeting domain / COOH, attached to the N-terminus of the HVEM extracellular domain. The leader sequence is a sequence used to induce expression of the adaptor protein in target cells and to release the adaptor protein extracellularly; it can also be omitted because the adaptor protein is only used to induce HSV infection of adjacent target cells after lysing the target cells and releasing HSV.

[0040] In this invention, for ease of cloning, when the scFv targeting the target molecule is used as a cell-targeting domain, the amino acid corresponding to any restriction enzyme site can be inserted between VH and VL; when the linker peptide is positioned between VH and VL, it can be inserted between VH or VL and the linker peptide; it can be inserted between scFv and HVEM; when the linker peptide is positioned between scFv and HVEM, it can be inserted between scFv and the linker peptide, or between the linker peptide and HVEM. For example, EF (base sequence: GAATTC) for restriction enzyme EcoRI or GS (base sequence: GGATCC) for BamHI can be inserted as shown in the following examples.

[0041] In this invention, to express single or any combination of factors to induce or enhance an immune response against cancer cells, recombinant HSV can be configured to insert the gene of the corresponding factor into the HSV genome. These factors can be manipulated to express cytokines, chemokines, immune checkpoint antagonists (e.g., antibodies, antibody derivatives, or antibody analogs, particularly scFv), co-stimulatory factors capable of inducing activation of immune cells (T cells or NK cells), antagonists capable of inhibiting the function of TGFβ, which suppresses the immune response against cancer cells (e.g., antibodies, antibody derivatives, or antibody analogs, particularly scFv), heparanases capable of degrading heparan sulfate proteoglycans in the solid tumor microenvironment, antagonists capable of inhibiting the function of the angiogenesis receptor VEGFR-2 (VEGF receptor-2) (e.g., antibodies, antibody derivatives, or antibody analogs, particularly scFv), and so on.

[0042] As cytokines, for example, interleukins such as IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, and IL-24, interferons such as IFNα, IFNβ, and IFNγ, tumor necrosis factor such as TNFα, and colony-stimulating factors such as GM-CSF and G-CSF can be used alone or in any combination of two or more of these cytokines for expression in recombinant HSV.

[0043] As chemokines, for example, CCL2 (CC motif chemokine ligand 2), CCL5 (RANTES), CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20 and XCL-1 (XC motif chemokine ligand 1) can be used alone or in combination for expression in recombinant HSV.

[0044] As immune checkpoint antibodies, antagonists against PD-1 (programmed cell death 1), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), CD27 (differentiation cluster 27), CD28 (differentiation cluster 28), CD70 (differentiation cluster 70), CD80 (differentiation cluster 80), CD86 (differentiation cluster 86), CD137 (differentiation cluster 137), CD276 (differentiation cluster 276), KIR (cytotoxic cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene 3), GITR (glucocorticoid-induced TNFR-related protein), GITRL (glucocorticoid-induced TNFR-related protein ligand), and CTLA-4 (cytolytic T lymphocyte-associated antigen-4) can be used alone or in combination for expression in recombinant HSV.

[0045] As costimulatory factors, CD2, CD7, LIGHT, NKG2C, CD27, CD28, 4-1BB, OX40, CD30, CD40, LFA-1 (lymphocyte function-associated antigen-1), ICOS (inducible T cell costimulator), CD3γ, CD3δ, and CD3ε can be used alone or in combination for expression in recombinant HSV.

[0046] In this invention, recombinant HSV can be manipulated to express a prodrug activating enzyme that converts a prodrug into a drug toxic to cancer cells. Examples of prodrug-activating enzymes may include cytosine deaminase, which converts 5-FC (5-fluorocytosine) as a prodrug into 5-FU (5-fluorouracil) as a drug; rat cytochrome P450 (CYP2B1), which converts CPA (cyclophosphamide) as a prodrug into PM (phosphoramide mustard) as a drug; carboxylesterase, which converts irinotecan (SN-38150) as a prodrug into SN-38 as a drug; bacterial nitroreductase, which converts BC1954 as a prodrug into 4-hydroxylamine 151 as a DNA cross-linking agent; PNP (purine nucleoside phosphorylase) isolated from Escherichia coli, which converts 6-methylpurine-2'-deoxyribonucleoside as a prodrug into 6-methylpurine as a drug, and so on.

[0047] Furthermore, in this invention, the recombinant HSV can be manipulated to express TRAIL (TNF-associated lysis-inducing ligand). TRAIL is known to induce cancer cell lysis by binding to its receptor, which is overexpressed in cancer cells (Kaoru Tamura et al., Multimechanistic Tumor-Targeted Oncolytic Virus Overcomes Resistance in Brain Tumors. Mol. Ther. 2013 Jan; 21(1):68-77).

[0048] For more details on the use of factors or prodrug activating enzymes to induce or enhance these immune responses, please refer to the following papers: [Michele Ardolino et al., Cytokine treatment in cancer immunotherapy, J. Oncotarget, Oncotarget. 14 Aug 2015; 6(23):], [Bernhard Homey et al., Chemokines: Agents for the Immunotherapy of Cancer, Nat Rev Immunol. 2002 Mar; 2(3): 175-84], [Marianela Candolfi et al., Evaluation of proapoptotic transgenes to use incombination with Flt3L in an immune-stimulatory gene therapy approach for Glioblastoma multiforme (GBM), J. FASEB] J., 2008, 22:107713], paper [Danny N Khalil et al. The Future of Cancer Treatment: Immunomodulation, CARs and Combination Immunotherapy. Nat Rev Clin Oncol. May 2016; 13(5):273-90], paper [Paul E Hughes et al. Targeted Therapy and Checkpoint Immunotherapy Combinations for the Treatment of Cancer. Trends Immunol. July 2016; 37(7):462-476], paper [Cole Peters, Samuel D. Rabkin. Designing herpes viruses as oncolytics. Mol. Ther Oncolytics. 2015; 2:15010], etc.

[0049] In this invention, as in the adaptor proteins described above, factors or prodrug activators that induce or enhance immune responses are configured to insert their gene expression cassettes (i.e., constructs in which their genes are operatively linked to promoter sequences and polyadenylation signal sequences that enable their expression) into the HSV genome without inhibiting HSV proliferation. This insertion can be performed without deletions in the HSV genome, or it can be inserted into loci where some or all of the non-essential genes in the HSV genome are missing. Here, when insertion occurs without deletions in the HSV genome, insertions can be made between genes; for example, preferred insertion loci are between UL3 and UL4 genes, between UL26 and UL27 genes, between UL37 and UL38 genes, between UL48 and UL49 genes, between UL53 and UL54 genes, and between US1 and US2 genes. When insertion occurs into loci where non-essential genes are missing, or within genes where non-essential genes are not missing, such non-essential genes can be selected from any of the non-essential genes described above.

[0050] Another aspect of the present invention relates to a pharmaceutical composition for anticancer treatment, which contains the above-mentioned recombinant HSV as an active ingredient.

[0051] The pharmaceutical compositions of the present invention have anticancer effects against tumors expressing a target molecule, which is targeted by the targeting domain of an adaptor protein expressed by recombinant HSV. Examples of tumors are described above with respect to the target molecule.

[0052] Specifically, preferably, the compositions of the present invention have anti-cancer effects against tumors with tumor cells expressing CEA or HER2. Examples of tumor cells expressing CEA include colorectal cancer cells, gastric cancer cells, lung cancer cells, breast cancer cells, rectal cancer cells, colon cancer cells, and liver cancer cells; examples of tumor cells expressing HER2 include breast cancer cells, ovarian cancer cells, gastric cancer cells, lung cancer cells, head and neck cancer cells, osteosarcoma cells, glioblastoma cells, and salivary gland tumor cells.

[0053] In this invention, the anticancer effects include dissolving cancer cells, reducing cancer cell activity, inhibiting or delaying the pathological symptoms of cancer by inhibiting cancer cell proliferation, inhibiting or delaying the onset of such pathological symptoms, inhibiting cancer metastasis, and inhibiting cancer recurrence.

[0054] In addition to recombinant HSV as the active ingredient, the pharmaceutical compositions of the present invention may also include recombinant adaptor molecules. A recombinant adaptor molecule means a fusion protein produced through a recombination process, said fusion protein having a cancer cell targeting domain as in the adaptor protein expressed by recombinant HSV, or more precisely, a cancer cell targeting domain as in a fusion protein of a cancer cell targeting domain and an HVEM extracellular domain. Here, having a cancer cell targeting domain as in the adaptor protein expressed by recombinant HSV means that when the cancer cell targeting domain of the adaptor protein expressed by HSV targets HER2, the cancer cell targeting domain of the adaptor molecule also targets HER2. Methods for producing target proteins of interest using recombination processes typically involve preparing an expression vector capable of expressing the target protein and transforming the expression vector into host cells such as E. coli, yeast, or animal cells (CHO cells, NSO cells, BHK cells, Sp2 cells, or HEK-293 cells), followed by culturing, and then isolating the target protein. Methods for producing target proteins of interest using recombination processes are well known in the art (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, (2001)). Specifically, regarding the generation of the recombinant linker molecules used in this invention, reference can be made to Korean Patent No. 10-0937774 and US Patent No. 8318662. The pharmaceutical compositions of this invention also include such recombinant linker molecules; therefore, when the recombinant HSV, which is the active ingredient of this invention, is administered to a patient together with the recombinant linker molecules, the initial infection efficiency of the recombinant HSV will be effectively improved.

[0055] Furthermore, the pharmaceutical compositions of the present invention can be used in combination with or as a mixture of approved anticancer agents. Examples of anticancer agents may include any anticancer agent that exhibits cytotoxicity against cancer cells, any cytokine drug, any antibody drug, any immune checkpoint inhibitor drug, and any cell therapy agent (for CAR-T cell therapy or CAR-NK cell therapy), such as metabolic antagonists, alkylating agents, topoisomerase antagonists, microtubule antagonists, and plant-derived alkaloids. Specific examples may include taxol and nitrogen mustard. Mustard, imatinib, oxaliplatin, gefitinib, bortezomib, sunitinib, carboplatin, cisplatin, rituximab, erlotinib, sorafenib, IL-2 drugs, IFN-α drugs, IFN-γ drugs, trastuzumab Examples of anticancer agents include tuzumab, blinatumomab, ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, bevacizumab, cetuximab, tisagenlecleucel (Kymriah), and axicabtagene ciloleucel (Yescarta). In addition to the exemplified anticancer agents, other anticancer agents known in the art may also be used (not limited to) in combination with or in mixtures with the pharmaceutical compositions of the present invention.

[0056] The pharmaceutical compositions of the present invention may include pharmaceutically acceptable carriers or excipients and are thus prepared into oral or parenteral formulations by typical methods known in the art, depending on the route of administration.

[0057] Pharmaceutically acceptable carriers or excipients of this type do not impair the activity or properties of the drug and are not toxic to humans themselves. Examples include lactose, dextran, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water (e.g., saline and sterile water), syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, Ringer's solution, buffers, maltodextrin solution, glycerol, ethanol, dextran, albumin, and any combination thereof. Specifically, when the pharmaceutical compositions of the present invention are formulated into liquid solutions, suitable carriers or excipients may include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextran solution, maltodextrin solution, glycerol, and ethanol, which may be used alone or in combination. If necessary, other typical pharmaceutical additives, such as antioxidants, buffers, and antibacterial agents, may be added and used.

[0058] When the pharmaceutical compositions of the present invention are prepared into oral formulations, they can be formulated into tablets, pills, capsules, elixirs, suspensions, syrups, rice paper, etc. When prepared into parenteral formulations, especially injections, they can be formulated into single-dose ampoules or multi-dose formulations. The pharmaceutical compositions of the present invention can also be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release formulations, etc.

[0059] The pharmaceutical compositions of the present invention can be formulated into unit dosage forms suitable for administration to a patient using typical methods in the pharmaceutical field, and can be administered via oral or parenteral routes using any administration method commonly used in the art, such as skin, intralesional, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transpulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, local, sublingual, intravaginal, and rectal routes.

[0060] The dosage (effective amount) of the pharmaceutical composition of the present invention may vary depending on factors such as: preparation method, administration mode, patient age, weight and sex, pathological state, diet, administration time, route of administration, excretion rate, and response sensitivity. These factors should be considered and determined by those skilled in the art. In a preferred embodiment, the pharmaceutical composition of the present invention is prepared as a unit dosage form injection. When prepared as a unit dosage form injection, the amount of recombinant HSV included per unit dose of the pharmaceutical composition of the present invention may be 10. 2 -10 14 PFU, especially 10 4 -10 11 Within the PFU range.

[0061] Another aspect of the present invention relates to a method for treating or preventing cancer (tumor), the method comprising administering to a subject, such as a patient, an effective amount of a pharmaceutical composition containing recombinant HSV as described above.

[0062] The method for treating cancer enables the treatment of cancer by dissolving and killing cancer cells that target molecules with the adaptor protein of recombinant HSV. Therefore, the treatment method of the present invention can be applied to any tumor having such target molecules. Specifically, the treatment method of the present invention is preferably applied to tumors expressing CEA or HER2.

[0063] The treatment methods of the present invention can be used (but are not limited to) in combination with other cancer treatment methods as described above. For example, cytotoxic anticancer agents, cytokine drugs, antibody drugs, immune checkpoint inhibitors, cell therapy agents (CAR-T cell therapy or CAR-NK cell therapy), radiotherapy, surgery, etc., can be used before or after the application of the compositions of the present invention, or in combination with the compositions of the present invention.

[0064] In the treatment methods of the present invention, the effective amount is the amount at which the pharmaceutical composition of the present invention, when administered to a subject such as a patient and continued for a period of time recommended by a medical expert, will cause the pharmaceutical composition of the present invention to exhibit the expected medical effect, such as cancer treatment or prevention. As mentioned above, such an effective amount should be determined by those skilled in the art, such as medical experts, based on the patient's age, weight and sex, pathological condition, etc., as described above.

[0065] In the treatment method of the present invention, the pharmaceutical composition is preferably administered in a parenteral mode, such as intralesional (tumor-bound), intravenous, intramuscular, or intra-arterial administration, or in the form of an injectable solution injected into the patient.

[0066] As described above, according to the present invention, a recombinant HSV containing an expression cassette capable of expressing a fusion protein of a cell-targeting domain and an HVEM extracellular domain can be provided; and its use can also be described.

[0067] When recombinant HSV infects target cells, i.e., cancer cells, and enters the target cells, HSV proliferates and expresses an adaptor protein, which acts as a fusion protein, within the cell. This adaptor protein is released extracellularly along with the proliferating HSV viral particles after cell lysis, or, if the adaptor protein contains a leader sequence, it can be released continuously even before the viral particles are released due to cell lysis. The extracellular fusion protein is used to induce HSV viral particles to infect surrounding cancer cells expressing target molecules recognized by cancer cell targeting domains, or to enhance infection efficiency.

[0068] Brief description of the attached diagram

[0069] Figure 1 The genomic structure of KOS-37BAC and the excision of BAC by the Cre-Lox system are illustrated schematically.

[0070] Figure 2 The genome structure of KOS-gD-R222N / F223I virus as HVEM-restricted HSV-1 is schematically shown;

[0071] Figure 3 The genome structure of KOS-EmGFP-gD-R222N / F223I virus expressing EmGFP as HVEM-restricted is schematically shown.

[0072] Figure 4 The results show the fluorescent expression of HSV-1 expressing EmGFP in HVEM-restricted cells and the outcome of specific infection of cells with HVEM receptors;

[0073] Figure 5 The genome structures of the KOS-HER2scFv-HveA-EmGFP-gD / R222N / F223I virus expressing the HER2scFv-HveA adaptor protein, the KOS-HveA-HER2scFv-EmGFP-gD / R222N / F223I virus expressing the HveA-HER2scFv adaptor protein, and the KOS-CEAscFv-HveA-EmGFP-gD / R222N / F223I virus expressing the CEAscFv-HveA adaptor protein are schematically shown.

[0074] Figure 6 The complete sequences and corresponding sequences of the HER2scFv-HveA adaptor protein, HveA-HER2scFv adaptor protein, and CEAscFv-HveA adaptor protein are shown.

[0075] Figure 7 The results of specific infection of HER2-expressing cells with KOS-HER2scFv-HveA-EmGFP-gD / R222N / F223I virus expressing HER2scFv-HveA adaptor protein are shown.

[0076] Figure 8 The results show the effects of specific lysis of HER2-expressing cells using KOS-HER2scFv-HveA-EmGFP-gD / R222N / F223I virus expressing the HER2scFv-HveA adaptor protein;

[0077] Figure 9The results show a comparison between lysing HER2-expressing cells with wild-type virus (HSV-1KOS) and KOS-HER2scFv-HveA-EmGFP-gD / R222N / F223I virus expressing HER2 adaptor protein (Her2 adaptor protein);

[0078] Figure 10 The results of CEA-expressing cells specifically infected with KOS-CEAscFv-HveA-EmGFP-gD / R222N / F223I virus expressing the CEAscFv-HveA adaptor protein are shown.

[0079] Figure 11 The results show a comparison between KOS-EmGFP-gD-R222N / F223I virus (gD / NI) expressing EmGFP-restricted HSV-1 and cells expressing CEA specifically infected with KOS-CEAscFv-HveA-EmGFP-gD / R222N / F223I virus expressing CEAscFv-HveA adaptor protein;

[0080] Figure 12 The results of specific infection with KOS-HER2scFv-HveA-EmGFP-gD / R222N / F223I virus expressing HER2scFv-HveA adaptor protein and KOS-HveA-HER2scFv-EmGFP-gD / R222N / F223I virus expressing HveA-HER2scFv adaptor protein are shown; and

[0081] Figure 13 The extracellular expression of the adaptor protein of the virus expressing the HER2scFv-HveA adaptor protein is shown, along with observations of viral infection spreading to surrounding cancer cells.

[0082] Description of specific embodiments

[0083] The invention will be better understood through the following examples. However, these examples should not be construed as limiting the scope of the invention.

[0084] <Example 1> Generation of HVEM-restricted HSV-1

[0085] The HSV-1 gene is a large gene of approximately 152 kb, therefore, KOS-37BAC (GenBank accession number MF156583) (Gierasch WW et al. J.Virol.Methods. 2006. 135:197-206) is used to insert foreign genes or introduce mutations into specific loci. The HSV-1 KOS strain is a primary HSV-1 strain used in the laboratory because of its well-known characteristics and suitability for studying gene function and pathogenesis (Smith KO. Proc. Soc. Exp. Biol. Med. 1964. 115:814-816). KOS-37BAC is prepared by inserting the BAC plasmid into the KOS gene and can be cloned at the bacterial level through transformation with DH10B bacteria (Invitrogen) (Gierasch WW et al.; J.Virol.Methods. 2006. 135:197-206). In KOS-37BAC, the bacterial artificial chromosome (BAC), along with flanking LoxP sites, is inserted into the locus between UL37 and UL38 of the HSV-1KOS genome. This was designed to allow for the removal of the BAC gene using the Cre-Lox system in a subsequent procedure. Figure 1 A schematic diagram is shown in the figure.

[0086] To produce HVEM-restricted HSV-1 (which enters cells only through the HVEM cell receptor), gD-R222N / F223IHSV-1 virus was produced, in which arginine (R) at position 222 and phenylalanine (F) at position 223 of the HSV-1 gD amino acid sequence (GenBank accession number ASM47818, SEQ ID NO:15) were replaced with asparagine (N) and isoleucine (I), respectively.

[0087] The gD-R222N / F223I HSV-1 virus produced by mutation can infect host cells only through HVEM (HveA) instead of cohesin-1 as the cell entry receptor (Uchida H et al., J.Virol. 2009. 83(7): 2951-2961).

[0088] Figure 2 The diagram schematically illustrates the genomic structure of HVEM-restricted HSV-1.

[0089] gD-R222N / F223I HSV-1 virus was produced by introducing the R222N / F223I mutation into the gD site of KOS-37BAC using an anti-selection BAC modification kit (GeneBridges) according to the manufacturer's protocol.

[0090] Specifically, *E. coli* clones containing KOS-37BAC were transformed with the pRed / ET plasmid, which expresses RecE and RecT, capable of performing homologous recombination (Muyrers JP et al.; Nucleic Acids Res. 1999. 27(6): 1555-1557). The gD-rpsL-neo / kan cassette was constructed using a set of homologous primers (forward primer gD-rpsL For: SEQ ID NO: 16, reverse primer gD-rpsL Rev: SEQ ID NO: 17), including a locus that introduces a mutation into gD. The gD-rpsL-neo / kan cassette consists of: the rpsL gene, which is a selective marker conferring streptomycin sensitivity; a gD homologous region at the insertion locus; and the neo / kan gene conferring kanamycin resistance. When the gD-rpsL-neo / kan cassette was inserted, *E. coli* strains sensitive to streptomycin via the rpsL gene and resistant to kanamycin via the neo / kan gene were created. *E. coli* strains were induced to express RecE and RecT by activating the function of pRed / ET through the addition of L-arabinose (Sigma-Aldrich) to *E. coli* clones containing KOS-37BAC and pRedET (Muyrers JP et al.; Nucleic Acids Res. 1999. 27(6): 1555-1557), after transformation with 200 ng of the prepared gD-rpsL-neo / kan cassette. The gD-rpsL-neo / kan cassette was inserted into the gD locus of KOS-37BAC via homologous recombination. *E. coli* strains with the gD-rpsL-neo / kan inserted into KOS-37BAC were resistant to kanamycin, but streptomycin resistance was blocked by the rpsL gene. This was the final step in selecting *E. coli* strains containing the inserted gD-rpsL-neo / kan gene from kanamycin-treated medium. After inducing RecE and RecT expression by activating pRed / ET function in *E. coli* containing the KOS 37-BAC gD-rpsL-neo / kan clone via the addition of L-arabinose (Sigma-Aldrich) to enable homologous recombination, the strain was transformed with the 100 pmol R222N_F223I_ mutant, an oligonucleotide encoding gD with N and I substitutions at positions 222 and 223 (SEQ ID NO: 18).Based on the principle of activating streptomycin resistance blocked by rpsL while replacing the existing gD-rpsL-neo / kan box with inserted oligonucleotides, candidates were selected in streptomycin medium (Heermann R. et al., Microb. CellFact. 2008. 14: doi: 10.1186). From the selected candidates, DNA was isolated using DNA preparation methods (Horsburgh BC et al., Methods Enzymol. 1999. 306: 337-352), and the N and I substitutions at positions 222 and 223 of gD were confirmed by PCR (polymerase chain reaction) and DNA sequencing.

[0091] Then, to generate the virus, intact KOS-BAC-gD-R222N / F223I DNA was extracted using a large construct DNA purification kit (Macherey-Nagel), followed by transfection with 1 μg of DNA at a rate of 2 × 10⁶ cells / mL using Lipofectamine 2000 reagent (Invitrogen). 5 Cre-Vero-HVEM cells were then cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 100 U / ml penicillin, 100 μg / ml streptomycin (Welgene), and 10% FBS (fetal bovine serum, Welgene). The Cre-Vero-HVEM cell line is a cell line that induces HVEM protein expression by inserting the HVEM gene into Cre-Vero cells (Gierasch et al.; J.Virol.Methods. 2006. 135: 197–206). The rationale for using Cre-Vero-HVEM is that the BAC gene of KOS 37BAC can be removed using the cell's Cre recombinase, and due to HVEM overexpression, it is effectively infected with KOS-gD-R222N / F223I virus, thus facilitating large-scale virus production. Three to four days after DNA transfection, spots were confirmed to form. Cells containing the virus were then collected and subjected to three freeze-thaw cycles (Gierasch WW et al.; J.Virol.Methods.2006.135:197-206) and sonicated to finally obtain the KOS-gD-R222N / F223I virus.

[0092] <Example 2> HSV-1 generation from HVEM-restricted expression of EmGFP

[0093] An expression cassette capable of expressing EmGFP (emerald green fluorescent protein) was inserted into the UL26 / UL27 locus of the KOS-gD-R222N / F223I virus fabricated in Example 1 above. EmGFP was used as a marker to facilitate the observation of viral generation and infection. The EmGFP cassette was fabricated using the pCDNA6.2-GW / EmGFP-miR plasmid (Invitrogen).

[0094] Figure 3 The diagram schematically illustrates the genomic structure of HSV-1 expressing UL26 / 27-EmGFP under HVEM restriction.

[0095] To express EmGFP, pCMV-EmGFP-tkpA, which uses the cytomegalovirus gene promoter and tkpA as the polyadenylation signal for HSV TK (herpes simplex virus thymidine kinase), was inserted into KOS-BAC-gD-R222N / F223I.

[0096] According to the manufacturer's instructions, all insertion methods were performed using the anti-selection BAC modification kit (GenBridges) as described in Example 1 above.

[0097] Specifically, *E. coli* clones containing the KOS-BAC-gD-R222N / F223I genome were transformed with the pRed / ET plasmid, which expresses RecE and RecT, capable of performing homologous recombination (Muyrers JP et al.; NucleicAcids Res. 1999. 27(6): 1555-1557). The UL26 / 27-rpsL-neo / kan cassette was constructed using a set of homologous region primers (forward primer UL26 / 27-rpsL_For: SEQ ID NO: 19, reverse primer UL26 / 27-rpsL_Rev: SEQ ID NO: 20), including the introduction of the target gene into the locus between UL26 and UL27. L-arabinose (Sigma-Aldrich) was added to clones containing KOS-BAC-gD-R222N / F223I and pRed / ET, thus inducing homologous recombination, followed by transformation with 200 ng of a UL26 / 27-rpsL-neo / kan cassette. Through homologous recombination, the UL26 / 27-rpsL-neo / kan cassette was inserted into the UL26 / 27 locus of KOS-BAC-gD-R222N / F223I. E. coli with the inserted UL26 / 27-rpsL-neo / kan cassette exhibited kanamycin resistance, but streptomycin resistance was blocked by the rpsL gene. This was presumed in E. coli selected from kanamycin-treated media containing the inserted UL26 / 27-rpsL-neo / kan cassette, and the final step of gene insertion was performed.

[0098] L-arabinose (Sigma-Aldrich) activating pRed / ET function was added to *E. coli* containing the UL26 / 27-rpsL-neo / kan cassette, thus inducing homologous recombination, followed by transformation with 200 ng of the UL26 / 27-tkpA-EmGFP-pCMV cassette. The UL26 / 27-tkpA-EmGFP-pCMV cassette was fabricated using pCDNA6.2-GW / EmGFP-miR plasmid (Invitrogen) as a template, forward primer UL26-tkpA_For (SEQ ID NO:21), and reverse primer UL27-pCMV_Rev (SEQ ID NO:22).

[0099] Based on the principle of activating streptomycin resistance blocked by rpsL while replacing the existing UL26 / 27-rpsL-neo / kan cassette with an inserted UL26 / 27-tkpA-EmGFP-pCMV, candidates were selected in streptomycin medium (Heermann R et al., Microb. Cell Fact. 2008. 14: doi: 10.1186). DNA was isolated from the selected candidates using DNA preparation methods (Horsburgh BC et al., Methods Enzymol. 1999. 306: 337-352). The introduction of tkpA-EmGFP-pCMV into UL26 / 27 was confirmed by EcoRI and XhoI treatment and PCR (polymerase chain reaction), and the exact gene sequence was identified by sequencing of the PCR product.

[0100] An experiment was conducted to investigate the normal expression of fluorescent proteins and viral generation. Intact KOS-BAC-EmGFP-gD-R222N / F223I DNA was extracted using a large construct DNA purification kit (Macherey-Nagel), followed by transfection of 2 × 10⁶ cells with 1 μg of DNA using Lipofectamine 2000 reagent (Invitrogen). 5 Cre-Vero-HVEM cells were transfected. Three days later, the fluorescence expression of EmGFP was observed using a fluorescence microscope, and virus production was observed through spot formation in Cre-Vero-HVEM cells. After confirming spot formation, virus-containing cells were collected, subjected to three freeze-thaw cycles (Gierasch WW et al.; J.Virol Methods. 2006. 135: 197–206), and then sonicated to obtain KOS-EmGFP-gD-R222N / F223I virus.

[0101] To infect KOS-EmGFP-gD-R222N / F223I virus and its fluorescent expression, HVEM-free cell lines (J1 and J-cohemin) and HVEM-expressing cell lines (J-HVEM) were used. J1 cells were juvenile hamster kidney cell lines lacking HVEM and cohemin-1, which are the viral HSV-1 receptors (Petrovic B. et al., 2017. PLoS Pathog. 19; 13(4):e1006352). J-cohemin and J-HVEM cell lines were cell lines that overexpressed cohemin-1 and HVEM, respectively, in J1 cells (Petrovic B. et al., 2017. PLoS Pathog. 19; 13(4):e1006352). Each cell line was cultured in DMEM (Welgene) containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS (fetal bovine serum, Welgene). 1 × 10⁻⁶ cells were cultured. 4 Cells were infected with the KOS-EmGFP-gD-R222N / F223I virus obtained above at 10 MOI (multiple of infection), and the expression of fluorescent protein and viral infection were observed using fluorescence microscopy after 24 hours (Baek HJ et al., Mol. Ther. 2011.19(3):507-514).

[0102] The result is Figure 4 As shown, the upper and lower images were acquired using fluorescence microscopy and optical microscopy, respectively. See also Figure 4 The upper fluorescence microscopy image shows that the J1 cell line and the J-adhesionin cell line were not infected, while only the J-HVEM cell line was infected.

[0103] Based on the above results, it is confirmed that, as expected, infection with KOS-EmGFP-gD-R222N / F223I virus can be easily observed through the expression of fluorescent proteins, and it is possible to use only HVEM, rather than cohesin-1, as the cell entry receptor for cell entry.

[0104] <Example 3> Generation of KOS-EmGFP-Gd-R222N / F223I virus expressing HER2scFv-HveA adaptor protein, HveA-HER2scFv adaptor protein, and CEAscFv-HveA adaptor protein.

[0105] Each of the boxes expressing HER2scFv-HveA adaptor protein, HveA-HER2scFv adaptor protein, and CEAscFv-HveA adaptor protein was inserted into the UL3 / UL4 locus of the KOS-EmGFP-gD-R222N / F223I virus manufactured in Example 2, in which the EmGFP expression box (tkpA-EmGFP-pCMV) was inserted.

[0106] Figure 5 The illustration schematically shows the KOS-HER2scFv-HveA-EmGFP-gD / R222N / F223I viral genome containing pCMV-HER2scFv-HveA-bGHpA as a cassette expressing the HER2scFv-HveA adaptor protein, the KOS-HveA-HER2scFv-EmGFP-gD / R222N / F223I viral genome containing pCMV-HveA-HER2scFv-bGHpA as a cassette expressing the HveA-HER2scFv adaptor protein, and the KOS-CEAscFv-HveA-EmGFP-gD / R222N / F223I viral genome containing pCMV-CEAscFv-HveA-bGHpA as a cassette expressing the CEAscFv-HveA adaptor protein. Furthermore, Figure 6 The complete sequences and corresponding sequences of the HER2scFv-HveA adaptor, HveA-HER2scFv adaptor, and CEAscFv-HveA adaptor are also shown. Here, the scFv of HER2 is configured as VH of SEQ ID NO:1 and VL of SEQ ID NO:2 linked via the linker peptide of SEQ ID NO:5, and the scFv of CEA is configured as VL of SEQ ID NO:3 and VH of SEQ ID NO:4 linked via the linker peptide of SEQ ID NO:6. In the HER2scFv-HveA adaptor and the CEAscFv-HveA adaptor, HveA is HveA82 of SEQ ID NO:7 excluding the leader sequence, and in the HveA-HER2scFv adaptor, it is Hve82 of SEQ ID NO:8 including the leader sequence. Similarly, in the HER2scFv-HveA adaptor and the CEAscFv-HveA adaptor, the leader sequence of SEQ ID NO:33 is included in the N-terminus, that is, before the VH of HER2scFv and the VL of CEAscFv.

[0107] Following the scFv sequence of HER2 or CEA and the NH2-GGGGS sequence (the linker sequence of the HveA sequence), EF (base sequence: GAATTC) is added. This is the EcoRI site for the easily cloned restriction enzyme. Following the HveA sequence and the NH2-GGGGS sequence (the linker sequence of the scFv sequence of Her2), GS (base sequence: GGATCC) is added. This is the BamHI site for the easily cloned restriction enzyme and is also a sequence that can be removed from the adaptor protein. bGHpA is the bGH-polyadenosine monophosphate (polyadenylation of bovine growth hormone) signal sequence. The full-length amino acid sequence and gene sequence of the HER2scFv-HveA adaptor protein used in this example are shown in SEQ ID NO:23 and SEQ ID NO:24, respectively; the full-length amino acid sequence and gene sequence of the HveA-HER2scFv adaptor protein are shown in SEQ ID NO:25 and SEQ ID NO:26, respectively; and the full-length amino acid sequence and gene sequence of the CEAscFv-HveA adaptor protein are shown in SEQ ID NO:27 and SEQ ID NO:28, respectively.

[0108] According to the manufacturer's instructions, insert the cassette expressing HER2scFv-HveA adaptor protein, the cassette expressing HveA-HER2scFv adaptor protein, and the cassette expressing CEAscFv-HveA adaptor protein using the anti-selection BAC modification kit (GeneBridges) as shown in Examples 1 and 2.

[0109] Specifically, *E. coli* clones containing the KOS-BAC-EmGFP-gD-R222N / F223I genome produced in Example 2 were transformed with the pRed / ET plasmid, which expresses RecE and RecT capable of performing homologous recombination (Muyrers JP et al.; Nucleic Acids Res. 1999. 27(6): 1555-1557). A set of homologous region primers (forward primer HSV-1_UL3 / 4-rpsL-neo_for: SEQ ID NO: 29, reverse primer HSV-1_UL3 / 4-rpsL-neo_rev: SEQ ID NO: 30) was used to construct the UL3 / 4-rpsL-neo / kan cassette, including introducing the target gene into the locus between UL3 and UL4. L-arabinose (Sigma-Aldrich) was added to clones containing KOS-BAC-EmGFP-gD-R222N / F223I and pRedET, thus inducing homologous recombination, followed by transformation with 200 ng of the more-prepared UL3 / 4-rpsL-neo / kan cassette. Through this type of homologous recombination, the UL3 / 4-rpsL-neo / kan cassette was inserted into the UL3 / 4 locus of KOS-BAC-EmGFP-gD-R222N / F223I. E. coli with the inserted UL3 / 4-rpsL-neo / kan cassette exhibited kanamycin resistance, but streptomycin resistance was blocked by the rpsL gene. This was presumed in the selection of E. coli with the inserted UL3 / 4-rpsL-neo / kan from kanamycin-treated media and was the final step in inserting the target gene.

[0110] L-arabinose (Sigma-Aldrich) was added to E. coli containing the UL3 / 4-rpsL-neo / kan box to activate the function of pRed / ET, thus inducing homologous recombination. Then, each of the following boxes was transformed with 200 ng of UL3 / 4-pCMV-Her2scFv-HveA-bGHpA, UL3 / 4-pCMV-HveA-Her2scFv-bGHpA, and UL3 / 4-pCMV-CEAscFv-HveA-bGHpA boxes. Using pCDNA3.1-HER2scFv-HveA plasmid, pCDNA3.1-HveA-HER2scFv plasmid, and pCDNA3.1-CEAscFv-HveA plasmid as templates, UL3 / 4-pCMV-Her2scFv-HveA-bGHpA cassettes, UL3 / 4-pCMV-HveA-Her2scFv-bGHpA cassettes, and UL3 / 4-pCMV-CEAscFv-HveA-bGHpA cassettes were fabricated using forward primer HSV-1_UL3 / 4-HM_pCMV_For (SEQ ID NO:31) and reverse primer UL3 / 4_bGH_poly_R (SEQ ID NO:32) (Baek HJ et al., Mol. Ther. 2011.19(3):507-514).

[0111] Based on the principle of activating streptomycin resistance blocked by rpsL and replacing the normally inserted UL3 / 4-rpsL-neo / kan cassette with the above-mentioned inserted UL3 / 4-pCMV-Her2scFv-HveA-bGHpA, UL3 / 4-pCMV-HveA-Her2scFv-bGHpA, and UL3 / 4-pCMV-CEAscFv-HveA-bGHpA, candidates were selected in streptomycin medium (Heermann R et al., Microb Cell Fact. 2008.14:doi:10.1186). DNA was isolated from the selected candidates using DNA preparation methods (Horsburgh BC et al., Methods Enzymol. 1999.306:337-352). Treatment with restriction enzymes EcoRI and XhoI, along with PCR (polymerase chain reaction), confirmed the introduction of UL3 / 4-pCMV-Her2scFv-HveA-bGHpA, UL3 / 4-pCMV-HveA-Her2scFv-bGHpA, and UL3 / 4-pCMV-CEAscFv-HveA-bGHpA into UL3 / 4. The exact gene sequences were identified by sequencing the PCR products.

[0112] Using a large construct DNA purification kit (Macherey-Nagel), intact KOS-BAC-Her2scFv-HveA-EmGFP-gD-R222N / F223I, KOS-BAC-HveA-Her2scFv-EmGFP-gD-R222N / F223, and KOS-BAC-CEAscFv-HveA-EmGFP-gD / R222N / F223I DNA was extracted. Then, 2 × 10⁶ cells were transfected with 1 μg of DNA using Lipofectamine 2000 reagent (Invitrogen). 5 Cre-Vero-HVEM cells were transfected. Three days later, the fluorescence expression of EmGFP and the formation of cell spots were observed using a fluorescence microscope. After confirming the formation of spots, virus-containing cells were collected, subjected to three freeze-thaw cycles (Gierasch WW et al.; J.Virol Methods. 2006. 135: 197–206), and then sonicated to obtain KOS-Her2scFv-HveA-EmGFP-gD-R222N / F223I, KOS-HveA-Her2scFv-EmGFP-gD-R222N / F223I, and KOS-CEAscFv-HveA-EmGFP-gD / R222N / F223I viruses.

[0113] <Example 4> Using oncolytic viruses expressing adaptor proteins to target cancer cells expressing HER2 or CEA.

[0114] <Example 4-1> Targeting HER2-expressing cancer cells with an oncolytic virus expressing the HER2scFv-HveA adaptor protein.

[0115] To evaluate whether the expression of the HER2scFv-HveA adaptor protein using the KOS-Her2scFv-HveA-EmGFP-gD-R222N / F223I virus manufactured in Example 3 induces viral infection of surrounding cancer cells or lysis after infection, the following experiments were performed.

[0116] The cell lines used in the experiment were non-HER2-expressing cell lines (J1, CHO-K1, MDA-MB-231) and HER2-expressing cell lines (J-HER2, CHO-HER2, SK-OV-3). Chinese hamster ovarian cell lines CHO-K1 and CHO-HER2 (Kuroki M et al., J. Biol. Chem. 1991. 74: 10132-10141) were cultured in HaM's F-12K medium (Welgene) containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS (fetal bovine serum), while J1 and J-HER2 (Petrovic B et al., 2017. PLoS Pathog. 19; 13(4): e1006352), breast cancer cell line MDA-MB-231 (ATCC, HTB-26) and ovarian cancer cell line SK-OV-3 (ATCC, HTB-77) were cultured in DMEM containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS.

[0117] For HER2-specific viral infections, 1×10 4 J cell line with 10 MOI, 1.5 × 10 4 CHO cell lines were used with 1 MOI and 1×10 4 SK-OV-3 and MDA-MB-231 cell lines were infected with the virus expressing the HER2scFv-HveA adaptor protein, produced in Example 3, at a MOI of 0.1. After 90 minutes, the medium was replaced with fresh medium to remove residual early virus and the HER2scFv-HveA adaptor protein. Viral infection in each cell line was observed by fluorescence microscopy by fluorescence expression 3 days after infection (Baek HJ et al., Mol. Ther. 2011.19(3):507-514).

[0118] The result is Figure 7 As shown, the images on the left and right sides were acquired using an optical microscope and a fluorescence microscope, respectively. See also Figure 7The fluorescence microscopy image on the right confirms that CHO-Her2, J-Her2, and SK-OV-3, as HER2-expressing cell lines, were specifically infected. The mechanism of the HSV-1 infection pathway involves the attachment of gB and gC to cells and entry into cells via gD. The reason why the gD R222N / F223I mutant virus expressing HER2scFv-HveA causes mild infection in the CHO-K1 cell line lacking HVEM and cohesin-1 is that, in addition to the function of gD, mild infection also occurs through cell attachment of gB and gC (Baek HJ et al., Mol. Ther. 2011.19(3):507-514).

[0119] Furthermore, as HER2-expressing cell lines, CHO-Her2, J-Her2, and SK-OV-3 were the first to be infected with the virus despite lacking the HVEM receptor. The reason for this is that the virus used for infection, which expressed the HER2scFv-HveA adaptor protein and was manufactured in Example 3, contained trace amounts of the HER2scFv-HveA adaptor protein that binds to viral gD, or the HER2scFv-HveA adaptor protein expressed during virus production in Cre-Vero-HVEM cells.

[0120] To observe the lysis caused by the virus, 1.5 × 10⁻⁶ 4 CHO-K1 and CHO-Her2 cell lines were infected with a virus expressing the HER2scFv-HveA adaptor protein at 1 MOI, and 1×10⁻⁶ cells were injected. 4 SK-OV-3 and MDA-MB-231 cell lines were infected with a virus expressing the HER2scFv-HveA adaptor protein at a 2 MOI. Each cell line was observed using an optical microscope 3 days after infection. Results were... Figure 8 As shown in the figure, in the case of CHO-Her2 and SK-OV-3, which are cell lines expressing HER2, the cell number can be seen to be much lower than that in the case of CHO-K1 and MDA-MB-231, which are cell lines not expressing HER2.

[0121] Furthermore, 1×10⁻⁶ cells will be used as the cell line expressing HER2. 4 SK-OV-3 cells were infected with each of HVEM-restricted herpesviruses (gD / NI) and viruses expressing the HER2scFv-HveA adaptor protein (Her2 adaptor protein) at 2 MOI, and lysis was observed on days 1, 2, 3, 4, and 5 using Alamar Blue (Sigma) cell staining. Results were... Figure 9As shown in the figure, the virus expressing the HER2scFv-HveA adaptor protein (Her2 adaptor protein) has a much higher effect on inducing lysis than the universal herpesvirus (HSV-1KOS).

[0122] <Example 4-2> Targeting CEA-expressing cancer cells with an oncolytic virus expressing the CEAscFv-HveA adaptor protein.

[0123] To evaluate whether expressing CEAscFv-HveA using the KOS-CEAscFv-HveA-EmGFP-gD / R222N / F223I virus expressing the CEAscFv-HveA adaptor protein manufactured in Example 3 induces viral infection of surrounding cancer cells, the following experiment was performed.

[0124] The cell lines used in the experiment were a CEA-free cell line (CHO-K1) and a CEA-expressing cell line (CHO-CEA, MKN45). The Chinese hamster ovary cell lines CHO-K1 and CHO-HER2 (Kuroki M et al., J. Biol. Chem. 1991. 74: 10132-10141) were cultured in HaM's F-12K medium (Welgene) containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS, and the gastric cancer cell line MKN45 (JCRB, JCRB0254) was cultured in RPMI-1640 medium containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS (Baek HJ et al., Mol. Ther. 2011. 19(3): 507-514).

[0125] Regarding CEA-specific viral infection, 1.5 × 10 4 CHO-K1 and CHO-CEA cells were infected with the virus at a MOI of 10. After 90 minutes, the culture medium was replaced with fresh medium to remove residual early virus and CEAscFv-HveA adaptor protein. After 72 hours, the degree of virus infection in each cell line was observed using a fluorescence microscope.

[0126] The result is Figure 10 As shown, the images on the left and right sides were acquired using an optical microscope and a fluorescence microscope, respectively. See also Figure 10 The fluorescence microscopy image on the right shows that the CHO-K1 cell line is rarely infected, while the CHO-CEA cell line is observed to be infected.

[0127] The reason why the gD R222N / F223I mutant virus expressing CEAscFv-HveA causes mild infection in CHO-K1 cell lines lacking HVEM and cohesin-1 is that, as mentioned above, in addition to the function of gD, mild infection also occurs through cell attachment of gB and gC (Baek HJ et al., Mol. Ther. 2011.19(3):507-514).

[0128] Furthermore, to confirm the specific infection of CEA-expressing cancer cells, 6 × 10 4 MKN45 cell lines were infected with 1 MOI of KOS-EmGFP-gD-R222N / F223I virus (gD / NI, control) prepared in Example 2 and KOS-CEAscFv-HveA-EmGFP-gD-R222N / F223I (scCEA-HveA) obtained in Example 3. After 90 minutes, the medium was replaced with fresh medium to remove residual early virus and CEAscFv-HveA adaptor protein. After 72 hours, the degree of virus infection in each cell line was observed using fluorescence microscopy.

[0129] The result is Figure 11 The images on the left and right sides are shown in the figure, acquired using optical microscopy and fluorescence microscopy, respectively. Referring to the image on the right, it can be seen that MKN45 cancer cells are specifically infected with the KOS-CEAscFv-HA-EmGFP-gD-R222N / F223I virus, but not with the KOS-EmGFP-gD-R222N / F223I virus used as a control.

[0130] <Example 5> The infectivity of oncolytic viruses expressing adaptor proteins varies depending on structural changes in the HER2 adaptor protein.

[0131] To evaluate whether the infectivity of the HER2scFv-HveA-EmGFP-gD-R222N / F223I virus expressing the HER2scFv-HveA adaptor protein, which was created in Example 3 with different adaptor protein structures, varies depending on the adaptor protein structure, the following experiments were performed.

[0132] The cell lines used in the experiment were the CHO-K1 cell line, which does not express HER2, and the CHO-HER2 cell line, which expresses HER2. The Chinese hamster ovary cell lines CHO-K1 and CHO-HER2 (Kuroki M et al., J. Biol. Chem. 1991. 74: 10132-10141) were cultured in HaM's F-12K medium (Welgene) containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS (fetal bovine serum).

[0133] To assess that viral infection activity depends on changes in adaptor protein structure, 1.5 × 10⁻⁶ mcg was used. 4 CHO-K1 and CHO-HER2 cell lines were infected with viruses expressing the HER2scFv-HveA adaptor protein and viruses expressing the HveA-HER2scFv adaptor protein, respectively, produced in Example 3, at 1 MOI. After 90 minutes, the culture medium was replaced with fresh medium to remove residual early virus and the HER2scFv-HveA and HveA-HER2scFv adaptor proteins. Three days after infection, cells were stained with VP16, a protein essential for early viral gene transcription, and the infection status of viruses expressing adaptor proteins with different structures was observed using fluorescence microscopy (Baek HJ et al., Mol. Ther. 2011.19(3):507-514).

[0134] See Figure 12 The image on the right, stained with VP16, shows the extent to which CHO-HER2 cells were infected with viruses expressing adaptor proteins of different structures. The infection levels of viruses expressing HER2scFv-HveA and HveA-HER2scFv adaptor proteins were similar. In the adaptor protein-expressing system, it was confirmed that the degree of infection did not differ based on the location of HveA, and that HER2-specific infectious activity was high for both structures. DAPI (4',6-diamidinyl-2-phenylindole) was used as a comparative indicator of VP16 staining by nucleic acid staining.

[0135] The reason why the gD R222N / F223I mutant virus expressing HER2scFv-HveA or HveA-HER2scFv causes mild infection in CHO-K1 cell lines lacking HVEM and cohesin-1 is that, as mentioned above, in addition to the function of gD, mild infection also occurs through cell attachment of gB and gC (Baek HJ et al., Mol. Ther. 2011.19(3):507-514).

[0136] Furthermore, as a HER2-expressing cell line, CHO-Her2 was the first to be infected with the virus despite lacking the HVEM receptor. The reason for this is that the virus used for infection, manufactured in Example 3 and expressing HER2scFv-HveA or HveA-HER2scFv adaptor proteins, contained trace amounts of HER2scFv-HveA or HveA-HER2scFv adaptor proteins that bind to viral gD, or HER2scFv-HveA expressed during virus production in Cre-Vero-HVEM cells.

[0137] <Example 6> Observation of extracellular expression of adaptor proteins in viruses expressing adaptor proteins and viral spread to surrounding cancer cells.

[0138] To confirm that the HER2scFv-HveA adaptor protein expressed by the KOS-Her2scFv-HveA-EmGFP-gD-R222N / F223I virus produced in Example 3 is released extracellularly, the following experiment was performed.

[0139] The cell line used in the experiment was the Vero-HVEM cell line (Gierasch et al.; J. Virol. Methods. 2006. 135: 197–206). The Vero-HVEM cell line was cultured in DMEM (Dalberg modified Eagle medium) containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS (fetal bovine serum, Welgene). 2.0 × 10⁻⁶ cells were cultured. 5 Vero-HVEM cells were infected with KOS-Her2scFv-HveA-EmGFP-gD-R222N / F223I virus and KOS-EmGFP-gD-R222N / F223I virus as a control at 0.1 MOI. After 90 minutes, the medium was replaced with fresh medium without FBS to remove residual early virus and HER2scFv-HveA adaptor protein. After 48 hours, the medium was collected. Protein expression levels were measured by Western blotting to confirm the expression of HER2scFv-HveA adaptor protein in the collected medium.

[0140] The result is Figure 13 As shown at the top. Figure 13The results at the top show that no expression of the adaptor protein was detected in the culture medium obtained from the group infected with KOS-EmGFP-gD-R222N / F223I virus (gD / NI) without the adaptor protein, but the adaptor protein released into the extracellular space was detected in the culture medium obtained from the group infected with KOS-Her2scFv-HveA-EmGFP-gD-R222N / F223I virus (Her2scFv-HveA).

[0141] Based on the above results, it is confirmed that, as expected, the expression of the inserted adaptor protein and its release into the extracellular space are effectively carried out through intracellular infection by the KOS-Her2scFv-HveA-EmGFP-gD-R222N / F223I virus.

[0142] In addition, to observe whether viral infection is due to cell lysis or the release of adaptor proteins into the extracellular space and spread to surrounding cancer cells, the following experiments were conducted.

[0143] The cell line used in the experiment was the SK-OV-3 cell line. As an ovarian cancer cell line, the SK-OV-3 cell line was cultured in DMEM (Dalberg modified Eagle medium) containing 100 U / ml penicillin / 100 μg / ml streptomycin (Welgene) and 10% FBS (fetal bovine serum, Welgene).

[0144] Then, 1×10 4 SK-OV-3 cell lines were diluted and infected with KOS-Her2scFv-HveA-EmGFP-gD-R222N / F223I virus at a MOI of 0.01. The reason for the viral dilution and infection was the observed spread of the virus to surrounding cancer cells. Viral infection and spread were observed using fluorescence microscopy.

[0145] The result is Figure 13 As shown at the bottom. Figure 13 The results at the bottom show that, 24 hours after infection, a group was observed to be infected with the KOS-Her2scFv-HveA-EmGFP-gD-R222N / F223I virus. Then, after 48 and 72 hours, rapid spread of the virus to surrounding cancer cells was observed in the first infection group.

[0146] Based on the above results, as expected, intracellular infection with KOS-Her2scFv-HveA-EmGFP-gD-R222N / F223I virus resulted in the release of inserted adaptor proteins extracellularly, which then bound to antigens on the surface of surrounding cancer cells. This confirmed the pattern of viral release due to cell lysis targeting adaptor proteins bound to cancer cells or using viral-bound adaptor proteins to spread infection to surrounding cancer cells expressing target molecules.

[0147] Although preferred embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various modifications, additions, and substitutions may be made without departing from the scope and spirit of the invention as disclosed in the appended claims. sequence list <110> Gisele Mead Company Korea Atomic Energy College <120> Recombinant herpes simplex virus with an expression cassette capable of expressing a fusion protein formed by a cancer cell targeting region and an HVEM extracellular domain and its uses <130> PF-B2725 <140> PCT / KR2020 / 011224 <141> 2020-08-24 <150> KR 10-2019-0103317 <151> 2019-08-22 <150> KR 10-2020-0072978 <151> 2020-06-16 <160> 33 <170> PatentIn version 3.5 <210> 1 <211> 120 <212> PRT <213> Artificial sequence <220> <223> HER2 VH <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> HER2 VL <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 3 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CEA VH <400> 3 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Asp 20 25 30 Gly Ile Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Phe Lys Tyr Met 35 40 45 Gly Trp Ile Asn Thr Ile Thr Gly Glu Pro Thr Tyr Thr Glu Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asp Glu Asp Thr Ala Thr Phe Phe Cys 85 90 95 Ala Lys Gly Thr Gly Thr Ser Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115 <210> 4 <211> 107 <212> PRT <213> artificial sequence <220> <223> CEA VL <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Ala Leu Ser Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Arg Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Asp Tyr Tyr Cys Gln His His Tyr Asn Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 15 <212> PRT <213> Artificial sequence <220> <223> HER2 linker <400> 5 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 6 <211> 14 <212> PRT <213> Artificial sequence <220> <223> CEA linker <400> 6 Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly 1 5 10 <210> 7 <211> 82 <212> PRT <213> Artificial sequence <220>e <223> HveA 82 <400> 7 Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys 1 5 10 15 Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu 20 25 30 Thr Gly Thr Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His 35 40 45 Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala 50 55 60 Met Gly Leu Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val 65 70 75 80 Cys Gly <210> 8 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Sp.‑HveA 82 <400> 8 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105 110 Arg Thr Glu Asn Ala Val Cys Gly 115 120 <210> 9 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> HveA87 <400> 9 Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys 1 5 10 15 Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu 20 25 30 Thr Gly Thr Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His 35 40 45 Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala [[ID= forty]]50 55 60 Met Gly Leu Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val 65 70 75 80 Cys Gly Cys Ser Pro Gly His 8⑤ <210> 10 <211> 125 <212> PRT <213> Artificial sequence <220> <223> Sp‑HveA87 <400> 10 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105 110 Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His 115 120 125 <210> 11 <211> 102 <212> PRT <213> Artificial sequence <220> <223> HveA102 <400> 11 Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys 1 5 10 15 Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu 20 25 30 Thr Gly Thr Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His 35 40 45[[ID=*15]] Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala 50 55 60 Met Gly Leu Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val 65 70 75 80 Cys Gly Cys Ser Pro Gly His Phe Cys Ile Val Gln Asp Gly Asp His 85 90 95 Cys Ala Ala Cys Arg Ala 100 <210> 12 <*211> 140 <212> PRT <213> Synthetic sequence <220> <223> Sp‑HveA102<* <400> 12 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro 1 5 10 15 * Note: There seems to be a potential error in the provided text as the tags <0* and <211> 140 have been marked with * for possible issues. If this is just a highlighting for review purposes and not actual errors, the translation remains as above. If they are indeed incorrect, the correct text should be provided for an accurate translation.Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105 110 Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys Ile 115 120 125 Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala 130 135 140 <210> 13 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> HveA107 <400> 13 Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys 1 5 10 15 Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu 20 25 30 Thr Gly Thr Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His 35 40 45 Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala 50 55 60 Met Gly Leu Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val[[ID=1十五]] 65 70 75 80 Cys Gly Cys Ser Pro Gly His Phe Cys Ile Val Gln Asp Gly Asp His 85 90 95 Cys Ala Ala Cys Arg Ala Tyr Ala Thr Ser Ser 100 105 <210> 14 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> Sp‑HveA107 <400> 14 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105 110 Arg Thr Glu Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys Ile 115 120 125 Val Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala Tyr Ala Thr Ser 130 135 140 Ser 145 <210> 15 <211> 369 <212> PRT <213> Artificial Sequence <220> <223> gD ASM47818 <400> 15 Lys Tyr Ala Leu Ala Asp Ala Ser Leu Lys Met Ala Asp Pro Asn Arg 1 5 10 15 Phe Arg Gly Lys Asp Leu Pro Val Leu Asp Gln Leu Thr Asp Pro Pro 20 25 30 Gly Val Arg Arg Val Tyr His Ile Gln Ala Gly Leu Pro Asp Pro Phe 35 40 45 Gln Pro Pro Ser Leu Pro Ile Thr Val Tyr Tyr Ala Val Leu Glu Arg 50 55 60 Ala Cys Arg Ser Val Leu Leu Asn Ala Pro Ser Glu Ala Pro Gln Ile 65 70 75 80 Val Arg Gly Ala Ser Glu Asp Val Arg Lys Gln Pro Tyr Asn Leu Thr 85 90 95 Ile Ala Trp Phe Arg Met Gly Gly Asn Cys Ala Ile Pro Ile Thr Val 100 105 110 Met Glu Tyr Thr Glu Cys Ser Tyr Asn Lys Ser Leu Gly Ala Cys Pro 115 120 125 Ile Arg Thr Gln Pro Arg Trp Asn Tyr Tyr Asp Ser Phe Ser Ala Val 130 135 140 Ser Glu Asp Asn Leu Gly Phe Leu Met His Ala Pro Ala Phe Glu Thr 145 150 155 160 Ala Gly Thr Tyr Leu Arg Leu Val Lys Ile Asn Asp Trp Thr Glu Ile 165 170 175 Thr Gln Phe Ile Leu Glu His Arg Ala Lys Gly Ser Cys Lys Tyr Ala 180 185 190 Leu Pro Leu Arg Ile Pro Pro Ser Ala Cys Leu Ser Pro Gln Ala Tyr 195 200 205 Gln Gln Gly Val Thr Val Asp Ser Ile Gly Met Leu Pro Arg Phe Ile 210 215 220 Pro Glu Asn Gln Arg Thr Val Ala Val Tyr Ser Leu Lys Ile Ala Gly 225 230 235 240 Trp His Gly Pro Lys Ala Pro Tyr Thr Ser Thr Leu Leu Pro Pro Glu 245 250 255 Leu Ser Glu Thr Pro Asn Ala Thr Gln Pro Glu Leu Ala Pro Glu Asp 260 265 270 Pro Glu Asp Ser Ala Leu Leu Glu Asp Pro Val Gly Thr Val Ala Pro 275 280 285 Gln Ile Pro Pro Asn Trp His Ile Pro Ser Ile Gln Asp Ala Ala Thr 290 295 300 Pro Tyr His Pro Pro Ala Thr Pro Asn Asn Met Gly Leu Ile Ala Gly 305 310 315 320 Ala Val Gly Gly Ser Leu Leu Ala Ala Leu Val Ile Cys Gly Ile Val 325 330 335 Tyr Trp Met His Arg Arg Thr Arg Lys Ala Pro Lys Arg Ile Arg Leu 340 345 350 Pro His Ile Arg Glu Asp Asp Gln Pro Ser Ser His Gln Pro Leu Phe 355 360 365 Tyr <210> 16 <211> 60 <212> DNA <213> Artificial sequence <220> <223> gD‑rpsL For <400> 16 cccaggccta ccagcagggg gtgacggtgg acagcatcgg gatgctgccc ggcctggtga 60 <210> 17 <211> 88 <212> DNA <213> Artificial sequence <220> <223> gD‑rpsL Rev <400> 17 ccggcgatct tcaagctgta tacggcgacg gtgcgctggt tctcggggat tcagaagaac 60 tcgtcaagaa ggcgtgatgg cgggatcg 88 <210> 18 <211> 369 <212> PRT <213> Artificial sequence <220> <223> gD R222N_F223I <400> 18 Lys Tyr Ala Leu Ala Asp Ala Ser Leu Lys Met Ala Asp Pro Asn Arg 1 5 10 15 Phe Arg Gly Lys Asp Leu Pro Val Leu Asp Gln Leu Thr Asp Pro Pro 20 25 30 Gly Val Arg Arg Val Tyr His Ile Gln Ala Gly Leu Pro Asp Pro Phe 35 40 45 Gln Pro Pro Ser Leu Pro Ile Thr Val Tyr Tyr Ala Val Leu Glu Arg 50 55 60 Ala Cys Arg Ser Val Leu Leu Asn Ala Pro Ser Glu Ala Pro Gln Ile 65 70 75 80 Val Arg Gly Ala Ser Glu Asp Val Arg Lys Gln Pro Tyr Asn Leu Thr 85 90 95 Ile Ala Trp Phe Arg Met Gly Gly Asn Cys Ala Ile Pro Ile Thr Val 100 105 110 Met Glu Tyr Thr Glu Cys Ser Tyr Asn Lys Ser Leu Gly Ala Cys Pro 115 120 125 Ile Arg Thr Gln Pro Arg Trp Asn Tyr Tyr Asp Ser Phe Ser Ala Val 130 135 140 Ser Glu Asp Asn Leu Gly Phe Leu Met His Ala Pro Ala Phe Glu Thr 145 150 155 160 Ala Gly Thr Tyr Leu Arg Leu Val Lys Ile Asn Asp Trp Thr Glu Ile 165 170 175 Thr Gln Phe Ile Leu Glu His Arg Ala Lys Gly Ser Cys Lys Tyr Ala 180 185 190 Leu Pro Leu Arg Ile Pro Pro Ser Ala Cys Leu Ser Pro Gln Ala Tyr 195 200 205 Gln Gln Gly Val Thr Val Asp Ser Ile Gly Met Leu Pro Asn Ile Ile 210 215 220 Pro Glu Asn Gln Arg Thr Val Ala Val Tyr Ser Leu Lys Ile Ala Gly 225 230 235 240 Trp His Gly Pro Lys Ala Pro Tyr Thr Ser Thr Leu Leu Pro Pro Glu 245 250 255 Leu Ser Glu Thr Pro Asn Ala Thr Gln Pro Glu Leu Ala Pro Glu Asp 260 265 270 Pro Glu Asp Ser Ala Leu Leu Glu Asp Pro Val Gly Thr Val Ala Pro 275 280 285 Gln Ile Pro Pro Asn Trp His Ile Pro Ser Ile Gln Asp Ala Ala Thr 290 295 300 Pro Tyr His Pro Pro Ala Thr Pro Asn Asn Met Gly Leu Ile Ala Gly 305 310 315 320 Ala Val Gly Gly Ser Leu Leu Ala Ala Leu Val Ile Cys Gly Ile Val 325 330 335 Tyr Trp Met His Arg Arg Thr Arg Lys Ala Pro Lys Arg Ile Arg Leu 340 345 350 Pro His Ile Arg Glu Asp Asp Gln Pro Ser Ser His Gln Pro Leu Phe 355 360 365 Tyr <210> 19 <211> 74 <212> DNA <213> Artificial sequence <220> <223> UL26 / 27-rpsL_For <400> 19 gcgtgggggg gaggaaatcg gcactgacca agggggtccg ttttgtcacg tcagaagaac 60 tcgtcaagaa ggcg 74 <210> 20 <211> 74 <212> DNA <213> Artificial sequence <220> <223> UL26 / 27-rpsL_Rev <400> 20 aacacataaa ctcccccggg tgtccgcggc ctgtttcctc tttcctttcc ggcctggtga 60 tgatggcggg atcg​​​​​​​​​​​​​​​​​​​​​​ <212> DNA <213> Artificial sequence <220> <223> UL27-pCMV_Rev <400> 22 aacacataaa ctcccccggg tgtccgcggc ctgtttcctc tttcctttcc tatacgcgtt 60 gacattgatt attg 74 <210> 23 <211> 351 <212> PRT <213> Artificial sequence <220> <223> HER2scFv-HveA adaptor <400> 23 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Gly Ala Arg Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly[[ID=4%]] 50 55 60 Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys 85 9% 95 Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 145 150 155 160 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 165 170 175 Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln 180 185 190 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe 195 200 205 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 210 215 220 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 225 230 235 240 Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly 245 250 255 Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Glu Phe Leu Pro Ser 260 265 270 Cys Lys Glu Asp Glu Tyr Pro Val Gly Ser Glu Cys Cys Pro Lys Cys 275 280 285 Ser Pro Gly Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu Thr Gly Thr 290 295 300 Val Cys Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His Leu Asn Gly 305 310 315 320 Leu Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala Met Gly Leu 325 330 335 Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val Cys Gly 340 345 350 <210> 24 <211> 1053 <212> DNA <213> Artificial sequence <220> <223> HER2scFv-HveA adaptor <400> 24 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacagg tgccagatgt 60 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 120 tcttgtgccg ccagcggctt caacatcaag gacacctaca tccactgggt ccgacaggcc 240. cctggcaaag gacttgaatg ggtcgccaga atctacccca ccaacggcta caccagatac gccgactctg tgaagggcag attcaccatc agcgccgaca ccagcaaga caccgcctac ctgcagatga acagcctgag agccgaggac accgccgtgt actactgttc tagaggggga 360 ggcgacggct tctacgccat ggattattgg ggccagggca ccctggtcac agttctagc 420 ggaggcggag gttctggcgg cggaggagt ggtggcggag gctctgatat ccagatgaca 480 cagagcccca gcagcctgtc tgcctctgtg ggagacagag tgaccatcac ctgtagagcc 540 agccaggacg tgaacacagc cgtggcttgg tatcagcaga agcctggcaa ggcccctaag ctgctgatct acagcgccag ctttctgtac agcggcgtgc ccagcagatt cagcggctct 660 agagcggca ccgacttcac cctgaccata agcagtctgc agccgagga cttcgccacc 720 tactactgtc to act caccacacct ccaaccttcg cagagggcac caaggtggaa 780 atcaagggtg gtggcggttc agaattcctg ccgtcctgca aggaggacga gtacccagtg 840 ggctccgagt gctgccccaa gtgcagtcca ggttatcgtg tgaaggaggc ctgcggggag 900 ctgacgggca cagtgtgtga accctgccct ccaggcacct acattgccca cctcaatggc 960 ctaagcaagt gtctgcagtg ccaaatgtgt gacccagcca tgggcctgcg cgcgagccgg1020 aactgctcca ggacagagaa cgccgtgtgt ggc 1053 <210> 25 <211> 369 <212> PRT <213> artificial sequence <220> <223> HveA‑HER2scFv adaptor <400> 25 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105 110 Arg Thr Glu Asn Ala Val Cys Gly Gly Gly Gly Gly Ser Gly Ser Glu 115 120 125 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr 145 150 155 160 Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 165 170 175 Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys 180 185 190 Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu 195 200 205 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser 210 215 220 Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly 225 230 235 240 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 245 250 255 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 260 265 270 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 275 280 285[[ID=!12]] Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 290 295 300 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val[[ID=!18]] 305 310 315 320 Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr 325 330 335 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 340 345 350 His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 355 360 365 Lys <210> 26 <211> 1107 <212> DNA <213> Artificial sequence <220> <223> HveA‑HER2scFv adaptor <400> 26 It should be noted that there are some tags marked with "!" in the translation which might need to be double - checked in the original context as they seem a bit unusual in this format. If they are just mis - marked in the original text you provided, they should be corrected according to the actual content.atggagcctc ctggagactg ggggcctcct ccctggagat ccccccag aaccgacgtc ttgaggctgg tgctgtatct caccttcctg ggagccccct gctacgcccc agctctgccg 120 tcctgcaagg aggacgagta cccagtgggc tccgagtgct gccccaagtg cagtccaggt 180 tatcgtgtga aggaggcctg cggggagctg acgggcacag tgtgtgaacc ctgccctcca 240 ggcacctaca ttgcccacct caatggccta agcaagtgtc tgcagtgcca aatgtgtgac ccagccatgg gcctgcgcgc gagccggac tgctccagga cagagaacgc cgtgtgtggc 360 ggtggtggcg gttcaggatc cgaggtgcag ctggttgaat ctggcggagg actggttcag 420 cctggcggat ctctgagact gtcttgtgcc gccagcggct tcaacatcaa ggacacctac 480 atccactggg tccgacaggc ccctggcaaa ggacttgaat gggtcgccag aatctacccc 540 accaacggct acaccagata cgccgactct gtgaagggca gattcaccat cagcgccgac accagcaaga acaccgccta cctgcagatg aacagcctga gagccgagga caccgccgtg tactactgtt ctagatgggg aggcgacggc ttctacgcca tggattattg gggccagggc 720 accctggtca cagtttctag cggaggcgga ggttctggcg gcggaggaag tggtggcgga 780 ggctctgata tccagatgac acagagcccc agcagcctgt ctgcctctgt gggagacaga 840 gtgaccatca cctgtagagc cagccaggac gtgaacacag ccgtggcttg gtatcagcag 900 aagcctggca aggcccctaa gctgctgatc tacagcgcca gctttctgta cagcggcgtg 960 cccagcagat tcagcggctc tagaagcggc accgacttca ccctgaccat aagcagtctg1020 cagcccgagg acttcgccac ctactactgt cagcagcact acaccacacc tccaaccttc1080 ggacagggca ccaaggtgga aatcaag 1107 <210> 27 <211> 346 <212> PRT <213> Artificial Sequence <220> <223> CEAscFv-HveA <400> 27 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn 35 40 45 Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro 50 55 60 Gln Leu Leu Val Tyr Asn Ala Lys Ala Leu Ser Glu Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Arg Ile Asn 85 90 95 Ser Leu Gln Pro Glu Asp Phe Gly Asp Tyr Tyr Cys Gln His His Tyr 100 105 110 Asn Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly 115 120 125 Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly Gln Ile Gln 130 135 140 Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys 145 150 155 160 Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Asp Gly Ile Asn 165 170 175 Trp Val Lys Gln Ala Pro Gly Lys Gly Phe Lys Tyr Met Gly Trp Ile 180 185 190 Asn Thr Ile Thr Gly Glu Pro Thr Tyr Thr Glu Asp Phe Lys Gly Arg 195 200 205 Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile 210 215 220 Asn Asn Leu Lys Asp Glu Asp Thr Ala Thr Phe Phe Cys Ala Lys Gly 225 230 235 240 Thr Gly Thr Ser Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 245 250 255 Ala Gly Gly Gly Gly Ser Glu Phe Leu Pro Ser Cys Lys Glu Asp Glu 260 265 270 Tyr Pro Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly Tyr Arg 275 280 285 Val Lys Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu Pro Cys 290 295 300 Pro Pro Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser Lys Cys Leu 305 310 315 320 Gln Cys Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala Ser Arg Asn 325 330 335 Cys Ser Arg Thr Glu Asn Ala Val Cys Gly 340 345 <210> 28 <211> 1038 <212> DNA <213> Artificial sequence <220> <223> CEAscFv‑HveA adaptor <400> 28 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacagg tgccagatgt 60 gacatccaga tgactcagtc tccagcctcc ctttctgcat ctgtgggaga cactgtcacc 120 atcacatgtc gagcaagtga gaacatttat agttatttag catggtatca gcagaaacag 180 ggaaaatctc ctcagctcct ggtctataat gcaaaggcct tatcagaagg tgtgccgtca 240 aggttcagtg gcagtggatc aggcacacag ttttctctga ggatcaacag cctgcagcct 300 gaagattttg gggattatta ctgtcaacat cattataatt ctccttatac gttcggaggg 360 gggaccaaac tggaaataaa gggctccacc tccgggtctg gtaaatcttc cgagggcaag 420 ggccagatcc agttggtgca gtctggacct gagctgaaga agcctggaga gacagtcaag 480 atctcctgca aggcttctgg ttattccttc acaaacgatg gaataaactg ggtgaagcag 540 gctccaggaa agggttttaa gtacatgggc tggataaaca ccatcactgg agagccaaca 600 tatactgaag acttcaaggg gcggtttgcc ttctctttgg aaacctctgc cagcactgcc 660 tatttgcaga tcaacaacct caaagatgag gacacggcta catttttctg tgcaaagggg 720 actgggacga gcgcttactg gggccaaggg actctggtca ctgtctctgc tggtggtggc 780 ggttcagaat tcctgccgtc ctgcaaggag gacgagtacc cagtgggctc cgagtgctgc 840 cccaagtgca gtccaggtta tcgtgtgaag gaggcctgcg gggagctgac gggcacagtg 900 tgtgaaccct gccctccagg cacctacatt gcccacctca atggcctaag caagtgtctg 960 cagtgccaaa tgtgtgaccc agccatgggc ctgcgcgcga gccggaactg ctccaggaca1020 gagaacgccg tgtgtggc 1038 <210> 29 <211> 79 <212> DNA <213> Artificial sequence <220> <223> HSV‑1_UL3 / 4‑rpsL‑neo_for <400> 29 taaataacac ataaatttgg ctggttgttt gttgtcttta atggaccgcc cgcaaggcct 60 ggtgatgatg gcgggatcg 79 <210> 30 <211> 78 <212> DNA <213> Artificial sequence <220> <223> HSV‑1_UL3 / 4‑rpsL‑neo_rev <400> 30 taggatcccg gccggatcgc gctcgtcacc cgacactgaa acgccccccc cccctcagaa 60 gaactcgtca agaaggcg 78 <210> 31 <211> 79 <212> DNA <213> Artificial sequence <220> <223> HSV‑1_UL3 / 4‑HM_pCMV_For <400> 31 taaataacac ataaatttgg ctggttgttt gttgtcttta atggaccgcc cgcaatatac 60 gcgttgacat tgattattg 79 <210> 32 <211> 78 <212> DNA <213> Artificial sequence <220> <223> UL3 / 4_bGH_poly_Rev <400> 32 taggatcccg gccggatcgc gctcgtcacc cgacactgaa acgccccccc ccccgcctca 60 gaagccatag agcccacc 78 <210> 33 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Signal sequence HER2 <400> 33 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Gly Ala Arg Cys 20

Claims

1. A recombinant herpes simplex virus, wherein an adaptor protein expression cassette expressing a fusion protein of a cancer cell targeting domain comprising an antibody and a HVEM extracellular domain is inserted into the genome of the herpes simplex virus without inhibiting the proliferation of the herpes simplex virus, the sequence of VH in the antibody is SEQ ID NO: 1 and the sequence of VL is SEQ ID NO: 2, or the sequence of VH is SEQ ID NO: 3 and the sequence of VL is SEQ ID NO: 4, wherein the sequence of the HVEM extracellular domain is selected from SEQ ID NO: 7 or 8, wherein the adaptor protein expression cassette is inserted between the UL3 and UL4 genes in the viral genome, and wherein the gene encoding glycoprotein D (gD) is mutated to prevent the encoded gD from binding to Nectin-1 while still retaining the ability of the gD to bind to HVEM.

2. The recombinant herpes simplex virus of claim 1, wherein the fusion protein is configured such that the cancer cell targeting domain is linked to the HVEM extracellular domain via a linker peptide comprising 1 to 30 amino acids.

3. The recombinant herpes simplex virus of claim 2, wherein the linker peptide comprises at least one amino acid selected from Ser, Gly, Ala, and Thr.

4. The recombinant herpes simplex virus of claim 1, wherein the cancer cell targeting domain is a domain that specifically recognizes and binds to HER2, which is a target molecule of the cancer cell that is the target cell, and the cancer cell targeting domain is a scFv, wherein the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2 are linked via a linker peptide in the scFv.

5. The recombinant herpes simplex virus of claim 4, wherein the linker peptide comprises the amino acid sequence of SEQ ID NO:

5.

6. The recombinant herpes simplex virus of claim 1, wherein the cancer cell targeting domain is a domain that specifically recognizes and binds to CEA, which is a target molecule of the cancer cell that is the target cell, and the domain is a scFv, wherein the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 4 are linked via the linker peptide in the order of VL, linker peptide, and VH.

7. The recombinant herpes simplex virus of claim 6, wherein the linker peptide comprises the amino acid sequence of SEQ ID NO:

6.

8. The recombinant herpes simplex virus of claim 1, wherein the recombinant herpes simplex virus is configured such that arginine (R) at position 222 and phenylalanine (F) at position 223 of the amino acid sequence of gD (glycoprotein D) are substituted with asparagine (N) and isoleucine (I), respectively.

9. The recombinant herpes simplex virus of claim 1, wherein the recombinant herpes simplex virus is a recombinant HSV-1 virus, a recombinant HSV-2 virus, or a HSV-1 and HSV-2 chimeric virus.

10. The recombinant herpes simplex virus of claim 1, wherein the recombinant herpes simplex virus is a recombinant HSV-1 derived from the HSV-1 KOS strain.

11. The recombinant herpes simplex virus of claim 1, wherein the recombinant herpes simplex virus is configured to further insert into the genome of the herpes simplex virus an expression cassette expressing any one selected from the group consisting of: (i) a cytokine; (ii) an immune checkpoint antagonist; (iii) a costimulatory factor that induces activation of immune cells; (iv) a TGF antagonist that suppresses immune response to cancer cells; (v) a heparanase that degrades heparan sulfate proteoglycans of a solid tumor microenvironment; (vi) an antagonist that suppresses function of the angiogenic receptor VEGFR-2 (VEGF receptor-2); and (vii) a prodrug-activating enzyme that converts a prodrug into a drug that is toxic to cancer cells, without suppressing proliferation of the herpes simplex virus.

12. The recombinant herpes simplex virus of claim 11, wherein the cytokine is a chemokine.

13. The recombinant herpes simplex virus of claim 11, wherein the cytokine is at least one selected from the group consisting of: IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-24, IFNa, IFP, IFNy, TNFa, GM-CSF, and G-CSF, the immune checkpoint is at least one selected from the group consisting of: PD-1 (programmed cell death receptor 1), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), CD276 (cluster of differentiation 276), KIR (killer cell immunoglobulin-like receptor), LAG3 (lymphocyte-activation gene 3), and CTLA-4 (cytotoxic T-lymphocyte-associated antigen-4), the costimulatory factor is at least one selected from the group consisting of: CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS (inducible T cell co-stimulator), and the prodrug-activating enzyme is at least one selected from the group consisting of: cytosine deaminase, rat cytochrome P450 2B1 (CYP2B1), carboxylesterase, bacterial nitroreductase, and PNP (purine nucleoside phosphorylase) isolated from E. coli.

14. The recombinant herpes simplex virus of claim 12, wherein the chemokine is at least one selected from the group consisting of: CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, and XCL-1.

15. The recombinant herpes simplex virus of claim 1, wherein the fusion protein is configured in the order of NH2 / cancer cell targeting domain / HVEM extracellular domain / COOH or in the reverse order.

16. The recombinant herpes simplex virus of claim 1, wherein the fusion protein is configured with the cancer cell targeting domain and the HVEM extracellular domain linked via a linking peptide, and the fusion protein is configured in the order NH2 / cancer cell targeting domain / HVEM extracellular domain / COOH or in the reverse order.

Images